BG105360A - Modulating multiple linease kinase proteins - Google Patents

Abstract

Methods for identifying compounds which modulate activity of a multiple lineage kinase protein and promotes cell survival or cell death comprising the steps of contacting the cell containing the multiple lineage kinase protein with the compound, determining whether the compound decreases activity of a multiple lineage kinase protein, and determining whether the compound promotes cell survival are provided. Method for identifying compounds which may be useful in the treatment of neurogenerative disorders and/or inflammation are also provided. Methods for modulating the activity of a multiple lineage kinase protein comprising contacting the protein of a cell containing the protein with an indeno- or indolo-compound of the invention are also provided. Methods of treating neurodegenerative disorders and/or inflammation are also provides. 135 claims, 23 figures

Description

FIELD OF THE INVENTION

The present invention is directed, in part, to methods for modulating members of the complex kinase family (MLK) family, methods for identifying compounds that modulate a complex kinase protein and promoting either cell survival or cell death, methods for determining compounds that may be useful in the treatment of neurodegenerative diseases and / or inflammation and methods of treating neurodegenerative diseases with compounds that inhibit a complex kinase protein line.

SUMMARY OF THE INVENTION

The MLK family consists of a group of proteins where the protein sequence of the kinase regions of the family members closely resembles MARKKK but have more similarity to each other than other MARKKK. The MLK family members contain some of the very complex kinase cascades, such as, for example, the stress signaling cascade, which involves modulating, among other things, the c-Jun N-terminus (JNK) kinase, which in turn modulates, among other things other, transcription factors including c-Jun, ATF2, and ELK-E JNK are described in US Patent Nos. 5,534,426, 5,593,884, 5,605,808, and WO 95/03324, each of which is hereby incorporated by reference in its entirety.

The MLK family includes, in turn, the following groups: 1) complex kinase line 1 (MLK1); 2) complex kinase line 2 (MLK2); 3) complex kinase line 3 (MLK3); 4) leucine zigzag kinase (LZK); 5) double leucine zigzag kinase (DLK); and 6) complex kinase line 6

(MLK6). MLK1 has a catalytic kinase region similar to kinases specific for both Tug and Ser / Thr. Dorow, et al., Eur. J. Biochem., 1993,213, 701-710. MLK2 also has a catalytic kinase region similar to kinases specific for both Tug and Ser / Thr. Dorow, et al., Eur. J. Biochem., 1993, 213, 701-710. MLK2 is also known as MST, Katoh, et al., Oncogene, 1995, 10, 1447-1451. MLK3 contains a protein that, in addition to the kinase region, contains two leucine zigzags with an adjacent carboxyl-terminal base region and a proline-rich region. Ing, et al., Oncogene, 1994, 9, 1745-1750. MLK3 is also known as SPRK (Gallo, et al., J. Biol. Chem., 1994, 269, 15092-15100) and PTK1 (Ezoe, et al., Oncogene, 1994, 9, 935-938). LZK is a leucine zigzag kinase. Sakuma, et al., J. Biol. Chem., 1997, 272, 28622-28629. The DLK has a kinase region and two putative leucine zigzag moving parts. Holzman, et al., J. Biol. Chem., 1994, 269, 30808-30817. DLK is also known as ZPK (Reddy, et al., Biochem. Biophys. Res. Comm., 1994, 202, 613-620) and MUK (Hirai, et al., Oncogene, 1996, 12, 641-650). Members of the MLK family are also described, for example, in US Patents 5,676,945, 5,554,523, WO 93/15201, Canadian Patent 2,148,898, Diener, et al., Proc. Natl. Acad Sci. USA, 1997, 94, 9687-9692, DeAizpurua, et al., J. Biol. Chem., 1997, 272, 16364-16373, Tung, et al., Oncogene, 1997, 14, 653659, Sells, et al., Trends in Cell Biol., 1997, 7, 161-167, Mata, et al. J. Biol. Chem., 1996, 271, 16888-16896, Hirai, et al., J. Biol. Chem., 1997, 272, 15167-15173, Fan, et al., J. Biol. Chem., 1996, 271, 24788-24793, Blouin, et al., DNA and Cell Biol., 1996, 75, 631-642, Pombo, et al., Nature, 1995, .377, 750-754, Kiefer, et al., EMBO J., 1996, 75, 7013-7025, Hu, et al., Genes & Dev., 1996, 10, 2251-2264, Su, et al., EMBO J., 1997, 16, 1279 -1290 and Dorow, et al, Eur. J. Biochem., 1995, 234, 492-500. Recently, another MLK-related kinase has been identified in the EST database. The DNA sequence of this clone, MLK6, has been described by seven matching messages. Their branch IDs are: 1007489,

1460085, 510915, 666323, F5555, 482188 and 178522, the sequences of all of which are incorporated herein by reference in their entirety.

Each of the references cited in this paragraph is incorporated herein by reference in its entirety.

Recently, the sustained expression of ZPK has been shown to reduce the proliferative capacity of NIH 3T3 fibroblasts as measured by the colony formation assay. Bergeron, et al., Biochem. Biophys. Really. Comm., 1997, 231, 153-155. However, Bergeron, et al., Failed to obtain any evidence demonstrating that ZPK modulates the activity of the ZPK substrate or that ZPK promotes cell death.

The expression of a structure encoding Myc-MLK2 in Swiss 3T3 cells has been shown to result in apoptosis approximately 20 hours after injection. Nagata, et al., EMBO J. 1998,17, 149-158.

Applicants have developed numerous indole and indeno compounds that, inter alia, inhibit cell growth associated with hyperproliferative conditions and inhibit death in various embryonic cultures, such as the dorsal root ganglion, the iliac body, the upper gingival cervical. Each of US Patent Nos. 5,475,110, 5,591,855, 5,594,009, 5,461,146, 5,621,100, 5,621,101, 5,705,511 and 5,756,494 is the basis of this application and each of these is incorporated herein by reference in its entirety. Compounds re-cited in U.S. Patent 5,705,511, of Formula G, relate to this application containing Formula I. Applicants have also shown that motoneuron apoptosis is inhibited by a derivative of K-252a, an indolecarbazole, which also modulates the stress signaling cascade . Maroney, et al., J. Neurosci., 1998, 18, 104-111, which is incorporated herein by reference in its entirety.

Due to the inadequacy of the study of compounds that modulate members of the stress signaling cascade and promote either cell death or cell survival, new, selective methods for the study of compounds continue to be required. In addition, tests are still needed to investigate drugs that can be used to treat inflammatory and neurodegenerative diseases. This study addresses these as well as other important end goals.

Summary of the invention

The present invention provides methods for identifying compounds that modulate complex kinase protein activity and promote cell survival, comprising the steps of contacting a cell containing a complex kinase protein with the compound, determining whether the compound reduces the activity of a complex kinase protein line and determining whether the compound promotes cellular survival.

The present invention also provides methods for identifying compounds that modulate complex kinase protein activity and promote cell death, comprising the steps of contacting a cell containing a complex kinase protein with the compound, determining whether the compound increases the activity of a complex kinase protein line and determining whether the compound promotes cell death.

The present invention also provides methods for identifying compounds that can be used in the treatment of neurodegenerative diseases consisting of contacting a cell or cell extract containing a complex kinase protein line with the compound and determining whether the compound reduces the activity of the complex kinase protein line .

The present invention also provides methods of identifying compounds that can be used in the treatment of inflammation, comprising contacting a cell or cell extract containing a complex kinase protein line with the compound and determining whether the compound reduces the activity of the complex kinase protein line.

The present invention also provides methods of treating mammals suffering or suspected of suffering from

a neurodegenerative disorder comprising administering to said mammal a compound that inhibits or reduces the activity of a complex kinase protein line.

The present invention also provides methods of treating mammals suffering from inflammation, comprising administering to said mammal a compound that inhibits or reduces the activity of a complex kinase protein line.

The present invention also provides methods of modulating the activity of a complex kinase protein line comprising contacting the protein or cell containing the protein with a compound of formula II:

wherein ring B and ring F, independently of each and together with the carbon atoms to which they are attached, are selected from the group consisting of:

Μβ unsaturated 6-membered carbocyclic aromatic ring, where from 1 to 3 carbon atoms can be replaced by nitrogen atoms;

an unsaturated 5-membered carbocyclic aromatic ring; and an unsaturated 5-membered carbocyclic aromatic ring, wherein either one carbon atom is replaced by an oxygen, nitrogen or sulfur atom;

two carbon atoms are replaced by a sulfur and nitrogen atom, an oxygen atom and a nitrogen atom or two nitrogen atoms; or three carbon atoms are replaced by three nitrogen atoms;

R ¹ is selected from the group consisting of:

H, substituted or unsubstituted alkyl having from 1 to 4 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heteroarylalkyl;

-C (= O) R ⁹ , wherein R ⁹ is selected from the group consisting of alkyl, aryl and heteroaryl;

-OR ¹⁰ , wherein R ¹⁰ is selected from the group consisting of H and alkyl having from 1 to 4 carbons;

-C (= O) NH ₂ , -NR ^h R ¹² , - (CH 2) _p NR ¹² R ¹² , - (CH 2) _p OR ¹⁰ , OO (CH 2) POR ¹⁰ and -O (CH 2) _p NR ⁿ R ¹² , wherein p is from 1 to 4; and where or

12

R and R are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbons; or

12

R and R together form a linking group is the formula - (CH 2 -X-CH 2 CH 2 -, wherein X ¹ is selected from the group consisting of -O-, -S- and CH ₂ -;

R is selected from the group consisting of H, alkyl having from 1 to 4 carbons, -OH, alkoxy having from 1 to 4 carbons, -OC (= O) R ⁹ , OC (= O) NR ⁿ R ¹² , - O (CH 2) _p NR ¹¹ R ¹² , -O (CH 2) _P OR ¹⁰ , substituted or unsubstituted arylalkyl having from 6 to 10 carbons and substituted or unsubstituted heteroarylalkyl;

R ³ , R ⁴ , R ⁵ and R ⁶ are each independently selected from the group consisting of:

H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF ₃ , -ΝΟ ₂ -, -OH, -OR ⁹ ,

-OiCH ^ pNR ¹¹ ^ ² , -OC (= O) R ⁹ , -OC (= O) NR ² R ⁷ , -OC (= O) NR ⁿ R ¹² , O (CH 2) pOR ¹⁰ , -CH 2 OR ¹⁰ , -NR ⁿ R ¹² , -NR ¹⁰ S (= O) 2 R ⁹ , -NR ¹⁰ C (= O) R ⁹ ; CH2OR ¹⁴ , where R ¹⁴ is an amino acid residue after the hydroxyl group of the carboxyl group is removed;

-NR ¹⁰ C (= O) NR ¹¹ R ¹² , -CO ² R ² , -C (-O) R ² , -C (= O) NR ⁿ R ¹² , -CH = NOR ² , CH = NR ⁹ , - ( CH 2) _p NR ⁿ R ¹² , - (CH ₂ ) _P NHR ¹⁴ or -CH = NNR ² R ^2A , where R ^2A is the same as R ² ;

-S (O) _y R ² - (CH 2) p S (O) _y R ⁹ , -CH 2 S (O) _y R ¹⁴ , where y is 0, 1 or 2;

alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons and alkynyl having from 2 to 8 carbons, wherein each alkyl, alkenyl or alkynyl group is unsubstituted; or each alkyl, alkenyl or alkynyl group is substituted by from 1 to 3 groups selected from the group consisting of aryl having from 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy, hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, C1-4 alkoxy, C1-4 , Br, I, -CN, -NO ₂ , -OH, -OR ⁹ , -X ² (CH 2) pNR ⁿ R ¹² , X ² (CH 2) _p C (= O) NR ⁿ R ¹² , -X ² ( CH 2) pOC (= O) NR ⁿ R ¹² , -X ² (CH 2) _p CO ₂ R ⁹ , X ² (CH 2) _p S (O) y R ⁹ , -X ² (CH 2) _p NR ¹⁰ C (= O) NR ⁿ R ¹² , -OC (= O) R ⁹ , -OCONHR ² , O-tetrahydropyranyl, -NR ⁿ R ¹² , -NR ¹⁰ C (= O) R ⁹ , -NR ¹⁰ CO ₂ R ⁹ ,

NR ¹⁰ C (= O) NR ⁿ R ¹² , -NHC (= NH) NH 2, NR ¹⁰ S (O) 2 R ⁹ , -S (O) y R ⁹ , -CO ² R ² , C (= O) NR ⁿ R ¹² , -C (= O) R ² , -CH ² OR ¹⁰ , -CH = NNR ² R ^2A , -CH = NOR ² , CH = NR ⁹ , -CH = NNHCH (N = NH) NH 2, -S (= O) ₂ NR ² R ^2A , -P (= O) (OR ¹⁰ ) ₂ , -OR ¹⁴ and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently or unsubstituted or replaced by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having 2 to 5 carbons, or alkoxy having 1 to 4 carbons;

X ² is O, S or NR ¹⁰ ;

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R and R are each independently selected from the group consisting of H, alkyl having from 1 to 4 carbons, alkoxy having from 1 to 4 carbons, substituted or unsubstituted arylalkyl having from 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl, - ( CH ₂ ) _P OR ¹⁰ , - (CH 2) pOC (^ O) NRR ¹² and (CH ₂ ) pNR R, or R and R together form a linking group of formula CH ₂ -X-CH ₂ -, where X is X or connection;

y and η are independently 0, 1 or 2;

Υ is selected from the group consisting of -O-, -S-, -N (R ¹⁰ ) -, -N ⁺ (O ') (R ¹⁰ ) -, N (OR ¹⁰ ) - and -CH ₂ -;

Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, C (= O) -, -CH (OR ¹⁰ ), -N (R ¹⁰ ) -, -N (OR ¹⁰ ) -, -CH (NR ⁿ R ¹² ) -, -C (= O) N (R ¹⁷ ) -, N (R ¹⁷ ) C (= O) -, -N (S (O) y R ⁹ ) -, -N (S (O) _y NR ⁿ R ¹² ) -, -N (C (= O) R ¹⁷ ) -, -C (R ¹⁵ R ¹⁶ ) -, -N ⁺ (O) (R ¹⁰ ) -, -CH (OH) -CH (OH) - and -CH (O (C = O) R ⁹ ) CH (OC (= O) R ^9A ) -, where R ^9A is the same as R ⁹ ;

R ¹⁵ and R ¹⁶ are independently selected from the group consisting of H, -OH, C (= O) R ¹⁰ , -O (CO) R ⁹ , hydroxyalkyl and -CO ₂ R ¹⁰ ;

R is selected from the group consisting of H, alkyl, aryl and heteroaryl;

A ¹ and A ² are selected from the group consisting of Η, Η; H, OR ² ; H, -SR ² ; H, -N (R) ₂ ; and a group where A and A taken together form a unit selected from the group containing = O, = S and NR ² ;

B ¹ and B ² are selected from the group consisting of Η, Η; H, -OR ² ; H, -SR ² , H, -N (R ² ) ₂ ; and a group wherein B ¹ and B ² together form a unit selected from the group containing = O, = -S and = NR ² ;

provided that at least one of the pairs A ¹ and A ² , or B ¹ and B ² , forms = 0.

The present invention also provides methods of modulating the activity of a complex kinase protein line comprising contacting the protein or cell containing the protein with a compound of formula III:

where

Zi is H and Ζ ₂ is H or Ζι and Z ₂ together form = 0;

R 1 is selected from the group consisting of H, Cl, CH ₂ SO ₂ C ₂ H ₅ , Br CH ₂ S (CH ₂ ) ₂ NH ₂ , CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH ₂ 11-C 4 H 9, NHCONHC ₆ H ₅ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ SC ₆ H ₅ , N (CH ₃ ) ₂ , CH 3, CH ₂ OCONHC ₂ H ₅ , NHCO ₂ CH ₃ , CH ₂ OC ₂ H ₅ , CH ₂ N (CH ₃ ) ₂ , OH, O-npropyl, CH = NNH-C (= NH) NH ₂ , CH = NN (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH-nC ₄ H ₉ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH ₃ ;

D \

CH = N-N NCH3 \ _7

R ₂ is selected from the group consisting of H, Br, Cl, I, CH ₂ S (CH ₂ ) 2 N (CH ₃ ) ₂ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH ₃ and CH ₂ OH;

X is selected from the group consisting of H, CH ₂ OH, CH ₂ NH-CepHHH, CO ₂ CH ₃ , CONHC ₆ H ₅ , CH ₂ NHCO ₂ C ₆ H ₅ , CH ₂ NHCO ₂ CH ₃ , CH ₂ N 3, CONHC ₂ H ₅ , CH ₂ MHT , CON (CH ₃ ) 2, -CH ₂ NHCO ₂ -, CONH ₂ , CONHC3H7, CH ₂ NH-CepHH, CH ₂ SOCH ₃ , CH = NOH, CH ₂ XH-Proline, CH ₂ CH ₂ (2-Pyridyl) , CH-NN HC (-NH) NH ₂ , CONH (CH ₂ ) ₂ OH, CH = NNHCONH ₂ , CH ₂ OCOCH ₃ , -CH ₂ OC (CH ₃ ) ₂ O-, CH ₂ SC ₆ H ₅ , CH ₂ SOC6H ₅ , CO ₂ n-hexyl, CONHCH ₃ and CO ₂ (CH ₂ ) ₄ CH ₃ ; or one of the following formulas

/ - \

CON O \ _ /

R is selected from the group consisting of OH and OCH ₃ .

The present invention also provides methods of modulating the activity of a complex kinase protein line comprising contacting the protein or cell containing the protein with a compound

of formula IV:

where

Zi is H and Z ₂ is H or Ζι and Z ₂ together form = 0;

R1 is H or Br;

R ₂ eH;

IV

R ₃ is H, CH ₂ CH = CH ₂ , CH ₂ CH ₂ CH ₂ OH or

R4 is H, CH ₂ CH = CH ₂ or CH ₂ CH ₂ CH ₂ OH.

Short description of the schemes

In order to illustrate aspects of the present invention, some features are shown in the diagrams. However, it should be known that this invention is not limited to the aspects shown.

Figure 1 is a schematic representation of the general preparation of bridged indenopyrrocarbazoles.

Figure 2 is a schematic representation of the general preparation of bridged indenopyrrolcarbazoles.

Figure 3 is a schematic representation of the preparation of indenopyrrolcarbazoles with synthetic resin as a binder.

Figure 4 is a schematic representation of the preparation of protected, soluble indenopyrrolcarbazoles.

Figure 5 is a schematic representation of the preparation of intermediate V.

Figure 6 is a schematic representation of the preparation of bridged indeno-pyrrolocarbazoles using method A.

Figure 7 is a schematic representation of the preparation of bridged indenopyrrolocarbazoles using method B.

Figure 8 is a schematic representation of the preparation of B-ring substituted bridged indenopyrrolocarbazoles.

Figure 9 is a schematic representation of the preparation of derivatives in the E ring of bridged indenopyrrolcarbazoles.

Figure 10 presents a graph of two separate experiments outlining the amount of viable neuronal differentiated PC-12 cells remaining after 5 days of culture in the absence of NGF. Results are presented as a percentage of control NGFs in each group (vehicle control in the absence of NGF, n = 12; all other groups, n = 3). The difference between media control and stable

cell samples expressing a dominant negative MLK-3 mutant in the absence of NGF were statistically significant, determined by a two-sided T-test (p <0.05).

Figure 11A shows the phosphorylation of GST-SEK-1 lifeless kinase by FLAG-MLK-3 (full length mixture and kinase region) extracted from rod virus using a radioactive gel based assay.

Figure II shows a ³² P-labeled phosphorylated myelin basic protein product resulting from a kinase reaction catalyzed by FLAG-MLK-3 (full-length and kinase blend) derived from rod virus or GST-MLK- 3 kinase region.

Figure 12 is an immunoassay showing the phosphorylation of GSTSEK-1 lifeless kinase by FLAG-MLK-3 (a mixture of full length and kinase region) derived from a rod virus determined by phosphospecific SEK-1 antibody.

Figure 13 is the phosphorylation of the myelin basic protein from a bacterially derived GST-MLK-3 kinase region using a (o) trifluoroacetic acid multiscreening test, or (a) phosphocellulose membrane method.

Figure 14 shows the saturation curve of [H] K252a incubated with lysate of insect cells infected with MLK-3 rod virus.

Figure 15 A shows the amount of ³² P-labeled c-jun in the immunoprecipitated / kinase response from MLK-3, MLK-2 or DLK over-expressing cells and treated with either 0.025% DMSO (control) or 500 nM K-252a.

Figure 15B shows a graphical quantification of the percentage activity remaining in immunoprecipitated / kinase responses from the samples described in Figure 15 A. The columns represent the average duplicate samples, where the error margin shows the variation in the mean.

Figure 15C shows the amount of β-labeled c-jun by immunoprecipitation / kinase response from cells that are overexpressing HA-JNK1 alone or with MEKK1 at different amounts of cellular DNA, as shown and treated or with 0.025% DMSO (control ) or 500 nM of Compound III-3 (see Table 3). The columns represent the average doubles, where the margin of error indicates the variation of the mean.

Figure 16 shows that Compound III-3 promotes neuronal survival in a concentration-dependent model. Dissociated neurons are cultured by sympathetic ganglia (SG) (A), dorsal root ganglia (DRG) (B), ciliary ganglia (CG) (C), and motoneurons (MN) (D) in the presence or absence of peat factors shown. Cells were enumerated 48 hours after insertion as described in materials and methods. Data represent mean ± SD of triple or quadruple determinations. One of three experiments is shown.

Figure 17 shows phase contrast micrographs of E12 DRG (A, E), E9 sympathetic (B, F), E8 ciliary (C, G) and E5.5 motor neurons (D, H) cultures after 48 hours in culture (24 hours for ciliary neurons) in the presence of the appropriate neurotropic factor (20 ng / mL NGF for sympathetic and sensory neurons 10 ng / mL CNTF for ciliary neurons, 30 pg / mL muscle extract (MEX) for motoneurons (A-D) or in the presence at 1 μΜ Compound III-E (E-H) Bar = 200 pm.

Figure 18 shows photomicrographs of dorsal root ganglion explants in vitro. DRG explants (E9) were placed in 96 well plates in medium containing 0.05% BSA. After a 2 hour attachment period, further: (A) control DMSO; (B) 20 ng / mL NGF; (C) 250 nM Compound III-3. Forty-eight hours later, the medium was removed and the explants were fixed with 4% paraformaldehyde in phosphate buffer solution.

Figure 19 shows the number of chicken lumbar motor neurons surviving on E10 after one day of treatment (E5-9) with specific doses of Compound III-3. Data presented is the mean ± SD of a group of 5-6 treated animals. This experiment is repeated twice. Data are from one representative experiment and represent one side of the lumbar column. * p <0.01, ** p <0.001, Student's t-test between Compound III-3 and Bonferroni-corrected control groups.

Figure 20 shows the number of motor neurons in the spinal nuclei of the tubercle cavernosus (SNB) of a female rat surviving on PN10 or PN60 after a one-day treatment (PN1-5) with Compound III-3, or control vehicle (5% Solution ™). On PN10 (A, B) or PN60 (B), rats were killed and the spinal cord region containing the SNB was repaired and prepared for histology; Cresylecht violet-stained motor neurons were then counted in a series of spinal cord slices at L5-S1 level, as described previously (Wingfield, et al., Steroids, 1975, 26, 311-327). The experimental data are mean ± S.E.M. from 4-8 animals per treatment group.

Figure 21 shows the loss of ChAT immunoreactivity after axonotomy of the sublingual nerve in the adult rat after treatment with Compound III-3. Photomicrographs of the nucleus of the sublingual nerve after incision and treatment with (A) filler solution (5% Solution ™) and (B) 200ug Compound III-3 applied from the incision side. (C) Number of ChAT immunoreactive motor neurons of the sublingual nerve after the treatment described in (A) and (B) above. The results are presented as the percentage of ChAT-immunoreactive motor neurons with 100% determined as the number of ChATimmunoreactive motor neurons in the contralateral uninjured nucleus of the sublingual nerve.

Figure 22 shows inhibition of the MLK-3 pathway demonstrates in vivo efficacy and blockade of phosphorylation at lower levels. Figure 22A shows an increase in tyrosine hydrolase of immunoreactive neurons of the black substance after a MPTP lesion with systemic administration of Compound III-3. Figure 22B is a representative immune staining showing MPTP causing an increase in levels of forforylated MKK4. Figure 22C outlines representative immune staining and an ELISA showing attenuation of MPTP-induced phosphorylated MKK4 in the presence of Compound III-3.

Figure 23 shows the induction of IL-2 in Jurkat cells. Figure 23A shows the time curve of IL-2 induction. Figure 23B shows the inhibition of IL-2 induction by Compound III-3. Figure 23C shows the inhibition of IL-2 induction by Compound III-3 and Compound 1-4.

Detailed description of the invention

The terms used above and throughout the description of the present invention, unless otherwise stated, are to be understood to have the following meanings.

"Apoptosis" refers to a specific morphological form of cell death characterized by the fragmentation of cells and their nuclei to particles enclosed by a membrane. Apoptosis can be induced, for example, by treatment with apoptosis-inducing compounds such as etoposide, staurosporine, tumor necrotic factor-α, ceramide and the like, or by conditions such as X-ray radiation.

The term "cell death" refers to the death of cells by apoptosis, necrosis, or other means commonly known to one of ordinary skill in the art. "Cell death" can be characterized, for example, as a decrease in the total number of cells or a decrease in cell viability compared to the untreated control cell population. Compounds that "promote cell death" lead to a decrease in cell number or cell viability compared to the control population. In contrast, compounds that "promote cell survival" lead to an increase in cell number or cell viability, or which delay or reduce the rate of cell death.

The terms "respond selectively" or "bind specifically" describes compounds that physically or chemically interact directly with an MLK protein. In contrast, compounds that "do not selectively respond" or "do not bind specifically" can affect proteins at lower or higher levels of MLK protein and thus may affect MLK protein activity but do not interact physically or chemically directly with the MLK protein.

The term &quot; modulates &quot; refers to an increase or decrease in the activity of a particular protein or substrate.

The present invention is directed, in particular, to methods of determining compounds that modulate MLK protein activity and promote either cell survival or cell death. Compounds that increase MLK protein activity can promote cell death, while compounds that decrease MLK protein activity may promote cell survival.

An MLK protein may be any protein designated as belonging to the MLK protein class. Preferably, the MLK protein is selected from the group consisting of MLK1, MLK2, MLK3, (SPRK, PTK1), LZK, DLK (ZPK, MUK) and MLK6, which are described above. In a preferred aspect of the invention, the methods identify compounds that directly interact with or bind to the MLK protein, as determined by binding studies, kinase assays, or other equivalent assays.

To identify compounds that modulate MLK protein activity and cause cell survival or cell death, a cell or cells containing MLK protein comes in contact with the test compound. The contact is made in buffers or media well known to those skilled in the art. Alternatively, the contact may be effected in vivo, wherein the animal, such as, for example, a mouse or other suitable animal known to those skilled in the art, is contacted by administering a pharmaceutical composition comprising the test compound and a pharmaceutically acceptable salt, carrier or diluent. In addition, different cell counts and concentrations of test compounds may be used. Determine whether the test compound increases or decreases the activity of the MLK protein. In addition, it is also determined whether the test compound promotes the cellular

survival or cell death.

Cells that are in contact with the test compounds can be cells of any mammal. Preferably, the cell is a neural cell. Preferably, the cell is involved in a neurodegenerative disease. For the purposes of this study, "neurodegenerative disease", "neurodegenerative disorder" and "neurodegenerative condition" are interchangeable and are used to describe any disease or disorder involving nerve cells or cells covered by the nervous system, including, but not limited to, limited only to, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntingtion disease and contusion or penetrating brain or spinal cord injuries.

MLK protein activity can be determined by various techniques. For example, MLK activity can be determined by measuring the activity of the MLK protein substrate. Such substrates are well known and readily apparent to those skilled in the art. Preferably, the substrate is a member of the mitogen family of kinases

activated protein kinase or family of kinases of mitogenically activated protein or further substrates, which include, but are not limited to, a protein selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρδγ, ρ38γ, ρ38γ, ρ38γ , MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and AEX-3 mammalian homologs, as well as common substrates of Ser / Thr protein kinases such as myelin basic protein (MBP). Reagents and methods for measuring substrate activity are also known to those skilled in the art. The presence of MLK can also be determined by measuring the amount of MLK protein or mRNA encoding the MLK protein. Reagents, including antibodies and oligonucleotide probes, as well as methods for measuring the amount of DNA or protein, including Northern and Western staining, are well known to those skilled in the art. MLK protein activity can also be determined by a kinase assay in vitvo. In vitro kinase assays are well known in the art. Other techniques for measuring protein activity are known to those skilled in the art and are encompassed by the present invention. Thus, one skilled in the art can determine whether the test compound modulates, i. increases or decreases the activity of the MLK protein.

Whether or not the test compound promotes cell survival or cell death is determined in several ways. It is preferable that the cause of cell survival or cell death is determined by using cells at risk of dying and comparing the amount of cells that have been in contact with the test compound and those remaining alive with the amount of cells that have not been in contact with test compound and other survivors. Preferably, the cells are primary embryonic cells of motoneurons that are programmed to die. Primary motoneuron embryonic cells have been described in Maroney, et al., 1998, 18, 104-111,

which is incorporated herein by reference in its entirety. Primary embryonic motoneuron cells will die unless they are released from the test compound. Thus, the higher number of living motoneuron cells in the population of motoneuron cells treated with the test compound, compared to the number of motoneuron cells in the population of motoneuron cells that were not treated with the test compound, is indicative of a test compound that promotes cell survival . In contrast, the smaller number of living motoneuron cells in the population of motoneuron cells treated with the test compound, compared with the number of motoneuron cells in the population of motoneuron cells that were not treated with the test compound, is indicative of a test compound that promotes cell death .

In another preferred aspect of the invention, normal cells or wild-type cells are converted to cells at risk of dying by overexpression of the MLK protein, as described in the Examples below, and then contacted with the test compound. . Cells that are overexpressing MLK proteins may die unless they are rescued by the test compound. The overexpression of MLK proteins can be performed using carriers that can express the protein within a cell. Expressive carriers are well known to those skilled in the art. In addition, methods for preparing expressive carriers are also known to those skilled in the art. Expression carriers that can express any of the MLK proteins can be prepared in a similar manner to those described in the Examples. The higher number of live cells in the overexpression cell population treated with the test compound, compared to the number of live cells in the over-expression cell population that was not treated with the test compound, is indicative of a test compound that promotes cell survival. Conversely, the smaller number of live cells in the overexpression cell population treated with the test compound compared to the number of live cells in the over-expression cell population that was not treated with the test compound is indicative of a test compound that

promotes cell death.

In another preferred aspect of the invention, promoting cell survival is determined by observing or measuring a decrease in apoptosis. Cytoplasmic contraction and nuclear condensation are associated with apoptosis. Thus, one skilled in the art can measure a decrease in apoptosis by measuring or observing a decrease in cytoplasmic contraction and / or nuclear condensation. In addition, one skilled in the art can measure apoptosis by applying conventional staining techniques.

In another preferred aspect of the invention, normal, wild-type neural cells can be used to identify compounds that promote cell death. Normal neural cells will survive unless their death is caused by the test compound. The smaller number of live cells in the normal cell population treated with the test compound compared to the number of live cells in the normal cell population that was not treated with the test compound is indicative of a test compound that promotes cell death. In contrast, the greater or equal number of live cells in the normal cell population treated with the test compound, compared to the number of live cells in the normal cell population that was not treated with the test compound, is not indicative of a test compound that assists cell death.

The present invention also relates, in part, to methods of modulating the activity of an MLK protein, comprising contacting the protein or cell containing the protein with a compound of formula G (designated Formula I) presented in US Patent No. 5,705,511, which has been transferred to the assignee of this application and incorporated herein by reference in its entirety.

The present invention also relates, in part, to methods of modulating the activity of an MLK protein consisting of contacting the protein or cell containing the protein with a compound of formula III presented below:

where

Z 1 is H and Z ₂ is H or Z 1 and Z ₂ together form = O;

R 1 is selected from the group consisting of H, Cl, CH ₂ SO ₂ C ₂ H ₅ , Br, CH ₂ S (CH ₂ ) ₂ NH ₂ , CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH ₂ , P-C 4 H 9, NHCONHC ₆ H ₅ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ SC ₆ H ₅ , N (CH ₃ ) ₂ , ch ₃ .

CH ₂ OCONHC ₂ H ₅ , NHCO ₂ CH ₃ , CH ₂ OC ₂ H ₅ , CH ₂ N (CH ₃ ) ₂ , OH, 0 p-propyl

CH = NNH-C (= NH) NH ₂ , CH = NN (CH ₃ ) ₃ , CH ₂ S (CH ₂ ) ₂ NH-nC ₄ H ₉ ,

CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH ₃ ,

R ₂ is selected from the group consisting of H, Br, Cl, I, CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH ₃ and CH ₂ OH;

X is selected from the group consisting of H, CH ₂ OH, CH ₂ NH-CepiniH, CO ₂ CH ₃ , CONHC ₆ H ₅ , ch ₂ nhco ₂ c ₆ h ₅ , ch ₂ nhco ₂ ch ₃ , ch ₂ n ₃ ,

CONHC ₂ H ₅ , CH ₂ NH-Fjihlihh. CON (CH ₃ ) ₂ , -CH ₂ NHCO ₂ -, CONH ₂ , CONHC3H7, CH ₂ NH-CepHH, CH ₂ SOCH ₃ , CH = NOH, CH ₂ CN-Proline,

CH ₂ CH ₂ (2-Pyridyl), CH = NNHC (= NH) NH ₂ , CONH (CH ₂ ) ₂ OH, ch = nnhconh ₂ , CH ₂ OCOCH ₃ , -CH ₂ OC (CH ₃ ) ₂ O-, ch ₂ sc ₆ h ₅ , CH ₂ SOC ₆ H ₅ , CO ₂ n-hexyl, CONHCH ₃ and CO ₂ (CH ₂ ) ₄ CH ₃ ; or one of the following formulas

/ - \ ^co1 \ _ /

and

R is selected from the group consisting of OH and OCH ₃ .

In a preferred aspect of the invention, Ζι and Z ₂ are Η, X is CO ₂ CH ₃ , R 1 is NHCONHC ₂ H ₅ , R ₂ is CH ₂ CH ₂ (2-Pyridyl) and R is OH. In other preferred aspects of the invention Ζι and Z ₂ are Η, X is CO ₂ CH ₃ , R 1 and R ₂ are CH ₂ OCH ₂ OCH ₂ CH ₃ and R is OH; or Z! and Z ₂ is Η, X is CO ₂ CH ₃ , R 1 and R ₂ are CH ₂ SCH ₂ CH ₃ and R is OH; or Z _b Z ₂ , R 1 and R ₂ are Η, X is CO ₂ CH ₃ ; and R is OH; or Z ₂ , Z ₂ , R 1 and R ₂ are Η, X is CO ₂ (CH ₂ ) ₄ CH ₃ and R is OH; or Z _b Z ₂ and R _b are H, R ₂ is CH ₂ OH, X is CO ₂ CH ₃ and R is OH; or Ζι and Ζ ₂ are H, R 1 and R ₂ are H ₂ 8 [3- (1,2,4-triazine)], X is CO ₂ CH ₃ and R is OH; or Ζι and Ζ ₂ are H, R 1 is Br, R ₂ is I, X is CO ₂ CH ₃ ; and R is OH; or Ζι and Z ₂ are H, R 1 and R ₂ are CH ₂ CH ₂ SCH ₃ , X is CO ₂ CH ₃ and R is OH; or Z _b Z ₂ , R 1 and R ₂ are Η, X is CO ₂ CH ₃ and R is OCH ₃ ; or Ζι and Ζ ₂ together form = O, R 1 and R ₂ are Br, X is CO ₂ CH ₃ and R is OH.

The present invention also relates, in part, to methods of modulating the activity of an MLK protein consisting of contacting the protein or cell containing the protein with a compound of formula II presented below:

wherein ring B and ring F, independently of each and together with the carbon atoms to which they are attached, are selected from the group consisting of:

(a) an unsaturated 6-membered carbocyclic aromatic ring in which 1 to 3 carbon atoms can be replaced by nitrogen atoms;

b) an unsaturated 5-membered carbocyclic aromatic ring; and

c) an unsaturated 5-membered carbocyclic aromatic ring in which either

1) one carbon atom is replaced by oxygen, nitrogen or sulfur atom;

2) two carbon atoms are replaced by a sulfur atom and a nitrogen atom, an oxygen atom and a nitrogen atom, or two nitrogen atoms; or

3) three carbon atoms are replaced by three nitrogen atoms; R ¹ is selected from the group consisting of:

a) H, substituted or unsubstituted alkyl of from 1 to 4 carbons, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl;

6) -C (= O) R ⁹ , wherein R ⁹ is selected from the group consisting of alkyl, aryl and heteroaryl;

b) OR ¹⁰ , wherein R ¹⁰ is selected from the group consisting of H and alkyl having from 1 to 4 carbons;

© r) -C (= O) NH ₂ , -NR ¹¹ R ¹² , - (CH2) pNR ¹¹ R ¹² , - (CH2) pOR ¹⁰ , (CH2) pOR ¹⁰ HO (CH2) pNR ⁿ R ¹² , where p is from 1 to 4; and where or

1) R and R are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbons; or

2) R ¹¹ and R ¹² together form a linking group of formula (CH 2b-XCHCH 2 X-, wherein X ¹ is selected from the group consisting of -O-, -S, and -CH ₂ -;

R is selected from the group consisting of H, alkyl having from 1 to 4 carbons, -OH, alkoxy having from 1 to 4 carbons, -OC (= O) R ⁹ , OC (= O) NR ⁿ R ¹² , - O (CH2) pNR ⁿ R ^12, -O (CH2) pOR ^10, substituted or unsubstituted arylalkyl © having from 6 to 10 carbons, and substituted or unsubstituted heteroarylalkyl;

R ³ , R ⁴ , R ⁵ and R ⁶ are each independently selected from the group consisting of:

a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF ₃ , -NO ₂ , -OH, -OR ⁹ , O (CH 2) pNR ⁿ R ¹² , -OC (= O) R ⁹ , -OC (= O) NR ² R ⁷ , -OC (= O) NR ⁿ R ¹² , O (CH 2) pOR ¹⁰ , -CH 2 OR ¹⁰ , -NR ⁿ R ¹² , -NR ¹⁰ S (= O) 2 R ⁹ , -NR ¹⁰ C (= O) R ⁹ ;

b) -CH ₂ OR ¹⁴ , where R ¹⁴ is the amino acid residue after the hydroxyl group of the carboxyl group is removed;

c) - NR ¹⁰ C (= O) NR ⁿ R ¹² , -CO 2 R ⁹ , -C (= O) R ² , -C (= O) NR ⁿ R ¹² , CH = NOR ² , -CH = NR ⁹ , - (CH 2) _p NR ⁿ R ¹² , - (CH ₂ ) _P NHR ¹⁴ , or -CH = NNR ² R ^2A where R ^2A is the same as R ² ;

r) -S (O) _y R ² - (CH 2) p S (O) _y R ⁹ , -CH 2 S (O) _y R ¹⁴ , where y is 0,1 or 2;

e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons and alkynyl having from 2 to 8 carbons, where

1) each alkyl, alkenyl or alkynyl group is unsubstituted;

or

2)) any alkyl, alkenyl or alkynyl group is

substituted by from 1 to 3 groups selected from the group consisting of aryl having from 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy, hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F, Cl, Br, I, -, CN, -NO ₂ , -OH, -OR ⁹ , X ² (CH 2) _p ^N R ⁿ R ¹² , -X ² (CH 2) _p C (= O) NR ⁿ R ¹² , -X ² (CH 2) pOC (= O) NR ⁿ R ¹² , X 2 (CH ₂ ) _P CO ₂ R ⁹ , -X ² (CH ₂ ) _p S (O) y R ⁹ , -X ² (CH ₂ ) _p N R ¹⁰ C (= O) NR ¹¹ R ¹² , OC (= O ) R ⁹ , -OCONHR ² , -O-tetrahydropyranyl, -NR ⁿ R ¹² , -NR ¹⁰ C (= O) R ⁹ , NR ¹⁰ CO2R ⁹ , -NR ¹⁰ C (= O) NR ¹¹ R ¹² , -NHC (= NH) NH 2, NR ¹⁰ S (O) ₂ R ⁹ , -S (O) y R ⁹ , -CO ² R ² , -C (= O) NR ⁿ R ¹² , -C (= O) R ² , -CH ₂ OR ¹⁰ , -CH = NNR ² R ^2A , -CHNOR ² , -CH = NR ⁹ , -CH = NNHCH (N = NH) NH 2, -S (= O) 2NR ² R ^2A , -P (= O) (OR ¹⁰ ) 2, -OR ¹⁴

and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently or unsubstituted or substituted by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons;

X ² eO, S, hbhNR ¹⁰ ;

8

R and R are each independently selected from the group consisting of H, alkyl having from 1 to 4 carbons, alkoxy having from 1 to 4 carbons, substituted or unsubstituted arylalkyl having from 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl, - (CH ₂ ) _P OR ¹⁰ , - (CH ₂ ) _p OC (= O) NR ⁿ R ¹² and 11 12 7 8 (CH ₂ ) _P NR R; or R and R together form a linking group of formula -CH ₂ -X ³ -CH ₂ -, wherein X ³ is X ² or a bond;

m and η are each independently 0.1 or 2;

Υ is selected from the group consisting of -O-, -S-, -N (R ¹⁰ ) -, -N ⁺ (O ') (R ¹⁰ ), -N (OR ¹⁰ ) -, and -CH ₂ -;

Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, C (= O) -, -CH (OR ¹⁰ ) -, -N (R ¹⁰ ) -, -N (OR ¹⁰ ) -, CH (NR ⁿ R ¹² ) -, -C (= O) N (R ¹⁷ ) -, N (R ¹⁷ ) C (= O) -, -N (S (O) yR ⁹ ) -, -N (S (O) yNR ⁿ R ¹² ) -, -N (C (= O) R ¹⁷ ) -, -C (R ¹⁵ R ¹⁶ ), -N ⁺ (O ') (R ¹⁰ ) - , -CH (OH) -CH (OH) - and -CH (O (C = O) R ⁹ ) CH (OC (= O) R ^9A ) -, where R is the same as R;

R ¹⁵ and R ¹⁶ are independently selected from the group consisting of H, -OH, -C (= O) R ¹⁰ , -O (C = O) R ⁹ , hydroxyalkyl and -CO ₂ R ¹⁰ ;

R is selected from the group consisting of H, alkyl, aryl and heteroaryl;

A ¹ and A ² are selected from the group consisting of Η, Η; H, OR ² ; H, -SR ² ;

H, -N (R) ₂ ,; and a group wherein A and A together form a unit selected from the group consisting of = O, = S and = NR ² ;

B ¹ and B ² are selected from the group consisting of Η, Η; H, -OR ² ; H, -SR ² ; H, -N (R ² ) ₂ ; and a group wherein B ¹ and B ² together form a unit selected from the group consisting of = O, = S and = NR ² ;

provided that at least one of the pairs A ¹ and A ² or B ¹ and B ² form = 0.

The present invention also relates, in part, to methods of modulating the activity of an MLK protein, comprising contacting the protein or cell containing the protein with a compound of formula IV, presented below:

where

Z 1 is H and Z ₂ is H or Z ₂ and Z ₂ together form = O;

R1 is H or Br;

R ₂ eH;

R ₃ is H, CH ₂ CH = CH ₂ , CH ₂ CH ₂ CH ₂ OH or CH ₂ CH ₂ CH ₂ -

R4 is H, CH ₂ CH = CH ₂ or CH ₂ CH ₂ CH ₂ OH.

In a preferred aspect of the invention,

R 1, R ₂ , R 4, Z 1 and Z ₂ are H and R ₃ is CH ₂ CH = CH ₂ . In another preferred aspect of the invention, R 1 is Br and R ₂ , R ₃ , R 4, and Z ₂ are H; or R _b R ₂ , Z 1 and Z ₂ are H and R ₃ and R 4 are CH ₂ CH = CH ₂ ; or R _b R ₂ , R ₃ , Z 1 and Z ₂ are H and R 4 is CH ₂ CH = CH ₂ ; or R _b R ₂ , Z 1 and Z ₂ are H and R ₃ and R 4 are CH ₂ CH ₂ CH ₂ OH; or R _b R ₂ , R 4, Z] and Ζ ₂ are H and R ₃ is

CH2CH2CH2— Ν Ο

The present invention also provides methods of identifying compounds that can be used in the treatment of neurological disorders, including contacting a cell or cell extract containing a complex kinase protein line with a

compound and determining whether the compound reduces the activity of a complex kinase protein line. The cells and the extracts obtained therefrom include those described above. Compounds found by the present methods (e.g., those compounds that inhibit or reduce the activity of a complex kinase protein) can be used to treat neurodegenerative disorders. The protein is preferably selected from the group consisting of complex kinase line 1, complex kinase line 2, complex kinase line 3, leucine zigzag kinase, double leucine zigzag kinase and complex kinase line 6. The cell contacts in vitro or in vivo. Preferably, the activity of the protein is determined by measuring the activity or degree of phosphorylation of the substrate of a given protein. Preferably, the substrate is selected from the group consisting of JNK1, JNK2, JNK3,

ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρ38δ, ΜΕΚ1, ΜΕΚ2, ICPC, ΜΚΚ4 (SEK1), ΜΕΚ5, ΜΚΚ6, ΜΚΚ7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3, as well as the common Ser / Thr, such as, for example, myelin basic protein (MBP). Protein activity can also be determined by measuring the activity of the protein substrate, the amount of protein substrate, or the mRNA encoding the protein substrate. Protein activity can also be determined by an in vitro kinase assay or binding assay. Cells are preferably primary embryonic motoneuron cells, cells that exhibit overexpression of a complex kinase protein or neural cell line, but may be any cell or cell extract. Preferably, compounds that bind directly to a complex kinase protein line are determined as described above.

The present invention also provides methods of identifying compounds that can be used in the treatment of inflammation, including contacting a cell or cell extract containing a complex kinase protein line with the compound and determining whether the compound reduces the activity of the complex kinase protein line. The cells and the extracts obtained therefrom include those described above. Compounds found by the present methods (e.g., those compounds that inhibit or reduce the activity of a complex kinase protein line) can be used to treat inflammation. The protein is preferably selected from the group consisting of complex kinase line 1, complex kinase line 2, complex kinase line 3, leucine zigzag kinase, double leucine zigzag kinase and complex kinase line 6. The cell contacts in vitro or in vivo. Preferably, the activity of the protein is determined by measuring the activity or degree of phosphorylation of the substrate of the protein. Preferably, the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρ38δ, MEK1, MEK2,

IPCC, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1 and a mammalian homolog of AEX-3, as well as common Ser / Thr substrates such as, for example, myelin basic protein (MBP). Protein activity can also be determined by measuring the activity of the protein substrate, the amount of protein substrate, or the mRNA encoding the protein substrate. Protein activity can also be determined by an in vitro kinase assay or binding assay.

Cells are preferably primary embryonic motoneuron cells, cells that exhibit overexpression of a complex kinase protein or neural cell line, but may be any cell or cell extract. Cells also include, but are not limited to, those involved in inflammation, such as, for example, lymphocytes, macrophages and other white blood cells, well known to those skilled in the art. Preferably, the compounds that directly bind a complex kinase protein line are identified.

The present invention also provides methods of treating mammals that have or are suspected to have a neurodegenerative disorder, including administering to the mammal a compound that inhibits or reduces the activity of a complex kinase protein line. A compound that inhibits or decreases the activity of a complex kinase protein line, but is not limited to, compounds of formula I, II, III and IV. Preferred compounds include those described above with respect to the method of investigating compounds that modulate the activity of a complex kinase protein line and promote cell survival or cell death. The preferred mammal is human. The individual may be a suspected neurodegenerative disease if he or she has symptoms of a particular neurodegenerative disease, is at high risk, or has a family history of neurodegenerative disease.

The present invention also provides methods of treating an mammal with inflammation, comprising administering to a mammal a

a compound that inhibits or reduces the activity of a complex kinase protein line. A compound that inhibits or decreases the activity of a complex kinase protein line includes, but is not limited to, compounds of formula I, II, III and IV. Preferred compounds include those described above with respect to the method of investigating compounds that modulate the activity of a complex kinase protein line and promote cell survival or cell death. The preferred mammal is human.

Contact with compounds of formula Ι-IV can be accomplished in buffers or media known to those skilled in the art. Alternatively, contacting may be effected by administering a pharmaceutical composition comprising the test compound and a pharmaceutically acceptable salt, carrier or solvent to a suitable animal or mammal, such as, for example, a mouse or other suitable animal known to those skilled in the art. In addition, different cell counts and compound concentrations may be used. The cells that contact the test compounds can be mammalian cells. Preferably, the cell is a neural cell. Preferably, the cell is involved in a neurodegenerative disease, such as, for example, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, epilepsy, Huntington's disease brain or spinal cord injuries. Compounds of formula I and methods for preparing the same are described in U.S. Patent 5,705,511, which is incorporated herein by reference in its entirety. Compounds of formula III and methods for preparing the same are described in US Patents 5,741,8098, 5,621,100, 5,621,101, 5,461,146 and 5,756,494 and WO 97/46567 and each is incorporated herein by reference in its entirety. Compounds of formula IV and methods for preparing them are described in US Patents 5,741,8098,

No. 5,621,100, 5,621,101, 5,461,146 and 5,756,494 and WO 97/46567 and each is incorporated herein by reference in its entirety.

Compounds of formula II include diastereomers and enantiomers around the carbon atoms to which the substituents R ² , R ⁷ h R ⁸ are attached.

Preferred bridged indenopyrrolocarbazoles are represented by Formula II:

Ri

P.

In preferred embodiments of the compounds of formula II, R 1 is H. In further preferred embodiments, R ² is H, hydroxide or substituted or unsubstituted alkyl.

In other preferred aspects, R ³ , R ⁴ , R ⁵ and R ⁶ are independently H, substituted or unsubstituted alkyl, halogen, substituted or unsubstituted alkoxy, substituted or unsubstituted amino or substituted or unsubstituted aryl. In further preferred embodiments, R ⁷ and R ⁸ are independently H, substituted or unsubstituted alkyl.

In some preferred embodiments, Y is O. In further preferred embodiments, Z is a bond, O, S, or substituted or unsubstituted N. In some still further preferred embodiments, m and η are independently 1 or 2. In some particularly preferred embodiments aspects, Υ is Ο, Ζ is a bond or O and m and η are independently 1 or 2.

12

In further preferred embodiments, AA and B are = O or H, H.

In some particularly preferred aspects, R ¹ , R ⁴ , R ⁶ and R ⁷ are each H, Y is = 0, η is 1, A ¹ A ² and B * B ² are = 0 or H, H, R ² is H, OH or lower alkyl, R ³ is H or substituted alkyl, R ⁵ and R ⁸ are each H or alkoxy, which is preferably methoxy, връзка is a bond or O and m is 1 or 2.

Some particularly preferred aspects of the compounds of formula II are compounds II-1, II-2, II-3, II-4a, II-4b, II-5, II-6, II-7a, II-7b, II-8 , P-9, ΠΙΟ, P-11 and P-12, shown below in Table 1.

The compounds represented by formula II are hereinafter referred to as Compound (II).

As used herein, the term &quot; carbocyclic &quot; refers to cyclic groups where the ring consists only of carbon atoms. The terms "heterocyclic" or "heterocyclic" refer to cyclic groups where the ring includes at least one heteroatom, such as Ο, N or S.

As used herein, the term "alkyl" means a straight-chain, cyclic or branched alkyl group having from 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, hexyl, octyl, cyclopropyl and cyclopentyl. The alkyl portion of the alkyl containing groups, such as alkoxy, alkoxycarbonyl and alkylaminocarbonyl groups, has the same meaning as the alkyl defined above. The lower groups that are preferred are the alkyd groups as defined above, which contain from 1 to 4 carbons. The term "alkenyl" is intended to include straight or branched chain hydrocarbons having at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl and propenyl groups. As used herein, the term "alkynyl" is intended to include straight or branched hydrocarbon chains having at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl and propynyl groups.

The acyl moiety of acyl containing groups, such as acyloxy groups, is intended to include straight or branched alkanoyl groups having from 1 to 6 carbon atoms, such as formyl, acetyl, propanoyl, butyryl, valeryl, pivaloyl or hexanoyl.

As used herein, the term "aryl" means a group having from 6 to 12 carbon atoms such as phenyl, biphenyl and naphthyl. Preferred aryl groups include unsubstituted or substituted phenyl and naphthyl groups. The term heteroaryl, as used herein, means an aryl group, wherein one or more ring carbon atoms are replaced by hetero (i.e., non-carbon) atoms, such as Ο, N or S. Preferred heteroaryl groups include pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl, triazolyl, tetrazolyl, quinolyl, isoquinolyl, benzoimidazolyl, thiazolyl, pyrazolyl and benzothiazolyl groups.

The term "aralkyl" (or "arylalkyl") is intended to mean a group having from 7 to 15 carbon atoms consisting of an alkyl group carrying an aryl group. Examples of aralkyl groups include benzyl, phenethyl, benzhydryl and naphthylmethyl groups.

Alkyl groups and alkyl units containing substituent groups such as aralkyl, alkoxy, arylalkoxy, hydroxyalkoxy, alkoxy-alkoxy, hydroxyalkylthio, alkoxyalkylthio, alkylcarbonyloxy, hydroxyalkyl and acyloxy groups may be substituted or substituted. A substituted alkyl group has from 1 to 3 independently selected substituents, preferably hydroxy, lower alkoxy, lower alkoxy-alkoxy, substituted or unsubstituted arylalkoxy-lower alkoxy, substituted or unsubstituted heteroarylalkoxy-lower alkoxy, substituted or unsubstituted or elbows.

arylalkoxy, substituted or unsubstituted heterocycloalkoxy, halogen, carboxyl, lower alkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, dioxolane, dioxane, dithiolane, dithion, furan, lactone

Ο

Substituted aryl, substituted heteroaryl and substituted aralkyl groups, each having from 1 to 3 independently selected substituents, which are preferably lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono- or di- lower alkylamino and halogen.

Heterocyclic groups formed by a nitrogen atom include pyrrolidinyl, piperidinyl, piperidino, morpholinyl, morpholino, thiomorpholino, N-methylpiperazinyl, indolyl, isoindolyl, imidazole, imidazolin, oxazolin, oxazol, triazolin, triazolin, triazolin, triazolin, triazolin, triazolin Heterocyclic groups formed by an oxygen atom include furan, tetrahydrofuran, pyran and tetrahydropyran groups.

"Hydroxyalkyl" groups are alkyl groups having a hydroxyl group attached thereto. Halogens include fluorine, chlorine, bromine and iodine.

As used herein, the term "heteroarylalkyl" means an arylalkyl group containing a heteroatom. The term "oxy" represents the presence of an oxygen atom. Thus, "alkoxy" groups are alkyl groups that are bonded through an oxygen atom and "carbonyloxy" groups are carbonyl groups that are bonded through an oxygen atom.

The term "heterocycloalkoxy" means an alkoxy group having a heterocyclo group attached to the derivative alkyl unit and the term "arylalkoxy" means an alkoxy group having an aryl group attached to the derivative alkyl unit. The term "alkylcarbonyloxy" means a group of formula -O-C (= O) -alkyl.

As used herein, the term "alkyloxy-alkoxy" means an alkoxy group containing an alkyloxy substituent attached to its alkyl moiety. The term "alkoxy-alkylthio" means an alkylthio group (i.e., a group of formula -S-alkyl) which contains an alkoxy substituent attached to its alkyl moiety. The term "hydroxyalkylthio" means an alkylthio group (i.e., a group of formula -S35

alkyl) which contains a hydroxy substituent attached to its alkyl unit.

As used herein, the term "monosaccharide" has the generally accepted meaning of simple sugar.

As used herein, the term "amino acid" means a molecule containing both an amino group and a carboxyl group. These include α-amino acids, i.e., carboxylic acids of the general formula HOOC-CH (No. 4 ₂ ) - (side chain).

The side chains of amino acids include naturally occurring and unnaturally occurring units. Unnaturally occurring (ie, unnatural) amino acid side chains are units that are used in place of naturally occurring amino acid side chains in, for example, amino acid analogs. See, e.g., Lehninger, Biochemistry, Second Edition, Worth Publishers, Inc., 1975, pp. 73-75, incorporated herein by reference.

Preferred α-amino acids include glycine, alanine, proline, glutamic acid and lysine having the D configuration, the L configuration or as a racemate.

The side chains of the further representative amino acids are shown below in Table 1.

Table 1

CH, HO-CHjOD-CH, HO-CJVCH _r

HS-CH, HOjC-CHCNHjhCHrS-S-CHjCHj-CH _r

CH ₃ -S-CH _r CH ₂ CH ₃ -CH _r S-CH ₂ -CH ₂ HO-CH ₂ -CH ₂ CHj-CH (OH> HO ₂ C-CH ₂ -NHC (= O) -CH, [ >

HOaC-CHj-CHjNH ₂ C (= O) -CH ₂ -CH ₂ (CH ₃ ) rCH (CH ₃ ) ₂ -CH-CH ₂ CH ₃ -CH ₂ -CH ₂ HjN-CHj-CHj-CHr H ₂ NC (= NH) -NH-CH ₂ -CH ₂ -CH ₂ H ₂ NC (= O) -NH-CH ₂ -CH ₂ -CH ₂ CH ₂ -CH ₂ -CH (CH ₃ > CH ₃ -CH ₂ -CH ₂ -CH ₂ H ₂ N-CH ₂ -CH ₂ -CH ₂ -CH ₂ B some preferred aspects, the substituent groups for the compounds of formula II include the amino acid residue after substitution of the hydroxyl group of the derivative carboxyl group, i.e. groups with formula -C (= O) -CH (MH ₂ ) - (side chain).

The functional groups present in the compounds of formula II may contain protecting groups. For example, the amino acid side-chain substituents of the compounds of formula II may be substituted by protecting groups such as benzyloxycarbonyl or Abutyloxycarbonyl groups. The protecting groups are known per se as functional groups that can be selectively linked to and substituted by functional groups such as hydroxyl and carboxyl groups. These groups are present in a compound to offer such functional inert to chemical reactions conditions to which the compound is exposed. Any variety of protecting groups can be used in the present invention. Such a protecting group is the benzyloxycarbonyl (Cbz; Z) group. Other preferred protecting groups of the invention can be found in Green, T.W. and Wuts, P.G.M., “Protective Groups in Organic Synthesis” 2d.Ed., Wiley & Sons, 1991.

Bridged indenopyrrolocarbazole compounds have clearly important functional pharmacological actions that find application in a variety of fields, including both research and therapeutic fields. These derivatives are used as therapeutic agents. The actions of the compounds show positive effects on the function and / or survival of the cells responsible for the trophic factor. The effect on the function and / or survival of the cells responsible for the trophic factor, i. neural row cells are indicated using any of the following tests: (1) spinal cord cholinacetyltransferase (ChAT) assay; or a test for the activity of cultured ChAT by basal forebrain neurons.

As used herein, the term "effect", when used to modify the terms "function" and "survival", means a positive or negative change or change. The effect that is

positive can be referred to here as "amplification" and an effect that is negative can be referred to here as "inhibition".

As used herein, the terms "enhancement" and "inhibition" when used to modify the terms "function" or "survival" mean that the presence of a bridged indenopyrrocarbazole compound has a positive effect on the function and / or survival of a cell responsible for trophic factor compared to cell in the absence of compound. For example, but not only regarding the survival of the so-called. cholinergic neuron, the compound will obviously enhance the survival of the cholinergic neural population at risk of death (as a result of, for example, trauma, disease, degenerative condition or natural progression) when compared to the cholinergic neural population without such compound if the treated population has a longer functional period than the untreated population.

As used herein, &quot; inhibit &quot; and &quot; inhibition &quot; mean that the specific response of a particular substance (e.g., enzyme activity) is relatively reduced in the presence of a bridged indenopyrrocarbazole compound.

As used herein, the term "trk" refers to a high affinity family of neurotrophic receptors, currently consisting of trkA, trkB, and trkC, and other membrane-bound proteins to which neurotrophin can bind.

As used herein, inhibition of VEGFR means use, for example, in diseases where angiogenesis, such as solid cancer tumors, endometriosis, diabetic retinopathy, psoriasis, hemangioblastoma, and other ocular diseases and cancers, plays an important role.

Inhibition of trk means use, for example, in prostate diseases such as prostate cancer

benign prostatic hyperplasia and treatment of pain from inflammation.

Inhibition of platelet-derived growth factor receptor (PDGFR) means use, for example in various forms of neoplasia, rheumatoid arthritis, pulmonary fibrosis, myelofibrosis, pathological wound healing, cardiovascular diseases such as atherosclerosis, resta and others.

As used herein, the terms "cancer" and "cancerous" refer to malignant cell proliferation in a mammal. Examples include prostate, benign prostatic hyperplasia, ovary, breast, brain, lung, pancreas, colorectum, stomach, solid tumors, head and neck, neuroblastoma, renal cell carcinoma, lymphoma, leukemia, other known malignancies, and other known malignant conditions. known cancers.

As used herein, the terms "neuron", "neural cell" and "neural cell" include, but are not limited to, a heterogeneous population of neural species having single or multiple transmitters and / or single or multiple functions; preferably, these are cholinergic and sensory neurons. As used herein, the phrase "cholinergic neuron" means Central Nervous System (CNS) Peripheral Nervous System (PNS) neurons whose neurotransmitter is acetylcholine; the basal forebrain, the brainstem in the brain, and the spinal cord neurons are exemplary. As used herein, the phrase "sensory neuron" includes neurons that are responsible for environmental irritants (eg, temperature, movement), such as skin, muscles and joints; dorsal root ganglion neuron is exemplified.

A "trophic factor responsible cell" as defined herein is a cell that includes a receptor to which the trophic factor can be specifically linked; examples include neurons (e.g.

cholinergic and sensory neurons) and non-neuronal cells (eg, monocytes and neoplastic cells).

The bridged indenopyrrolocarbazole compounds described herein find application in both research and therapeutic fields, for example, in inhibiting enzyme activity. For example, in the research field compounds can be used in the development of tests and models to further facilitate the understanding of the role that inhibition of the serine / threonine or tyrosine protein kinase (e.g., PKC, trk tyrosine kinase) plays in the mechanical aspects related disorders and diseases. In the therapeutic field, compounds that inhibit this enzyme activity can be used to inhibit the deleterious effects of these enzymes on disorders such as cancer.

As the examples below show, inhibition of enzyme activity using bridged indenopyrrocarbazole compounds can be determined using, for example, the following tests:

1. trkA Test for inhibition of tyrosine kinase activity;

2. Inhibition of NGF-stimulated trk phosphorylation in whole cell preparation;

3. Vascular endothelial growth factor receptor (VEGFR) kinase inhibition assay;

4. PKC activity inhibition test;

5. PDGFR inhibition test.

The disclosed bridged indeno-pyrrolocarbazole compounds can be used to accelerate the function and / or survival of mammalian neural row cells, e.g. man. In this sense, the compounds may be used individually or with other fused pyrrolocarbazoles and / or indolocarbazoles or in combination with other advantageous molecules that also demonstrate the ability to affect the function and / or survival of a cell.

A large number of neurological disorders are characterized by neuronal cells that have died, are injured, are functionally incapacitated, have suffered axonal degeneration, are at risk of dying, etc. These disorders include, but are not limited to, Alzheimer's disease, motor neuron disorders (e.g., amyotrophic lateral sclerosis); Parkinson's disease; cerebrovascular disorders (eg stroke, ischemia); Huntington's disease; AIDS dementia; epilepsy; multiple sclerosis; peripheral neuropathies (for example, those that affect DRG neurons in peripheral chemotherapy-related neuropathy) including diabetic neuropathy; disorders caused by excitatory amino acids; and disorders related to contusional or penetrating digestive tract of the brain or spinal cord.

ChAT catalyzes the synthesis of neurotransmitter acetylcholine and is thought to be an enzyme marker for functional cholinergic neuron. A functional neuron can also survive. Neural survival is investigated by quantifying specific uptake and enzymatic conversion of dye (eg, calcein AM) by living neurons.

Due to their various uses, the compounds described herein, including those compounds identified by the methods described herein, find application in various fields. The compounds may be used in the development of in vitro models of neural cell survival, function, identification, or for the study of other synthetic compounds that have activities similar to those of the compounds described herein, or compounds determined by the methods described herein. The compounds described herein, as well as those determined using the methods described herein, can be used in a research area to detect, delineate and identify molecular targets associated with functional responses. The entire target to which the derivative binds can be identified, isolated and purified for characterization, for example by X-ray labeling of an indenopyrrolocarbazole compound or

a compound defined by methods described herein related to a specific cellular function (e.g., mitogenesis).

Compounds, both those described herein and those determined using the methods described herein, are used not only to accelerate the action of trophic cells, e.g., cholinergic neurons, by the trophic factor, but may also act as survival agents for other neural cell types, eg, dopaminergic or glutamatergic. Growth factor can regulate neuronal survival by signaling a cascade chain of small GTP binding proteins, which include, but is not limited to, ras, kerosene and cdc42 (Denhardt, Biochem. J., 1996, 318, 729). Specifically, ras activation leads to phosphorylation and activation of extracellular receptor-activated kinase (ERK), which is associated with biological growth and differentiation processes. Stimulation of rac / cdc42 leads to increased activation of JNK and p38, responses that are associated with stress, apoptosis and inflammation. Although growth factor responses are initially in the ERK pathway, interference with the latter processes may lead to alternative mechanisms of neural survival, which may resemble the properties of growth factor that accelerates survival (Xia et al., Science, 1995, 270 , 1326). The compounds may also function as survival agents for neural and non-neuronal cells through mechanisms that relate, but not only, to survival via growth factor, for example, inhibition of the JNK pathway and p38, which may lead to survival by inhibiting the processes of apoptotic cell death.

The present compounds are useful in the treatment of disorders associated with decreased ChAT activity or death, spinal cord motoneuron injury, and also used, for example, in diseases associated with apoptotic cell death of central and

peripheral nervous system, immune system and inflammatory diseases.

The compounds described herein may also find application in the treatment of disease states involving malignant cell proliferation, such as many cancers.

The pharmaceutically acceptable salts of the compounds described herein, as well as those compounds determined by the present methods, include pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts and amino acid addition salts. Examples of acid addition salts are inorganic acid addition salts such as hydrogen chloride, sulfate and phosphate and organic acid addition salts such as acetate, maleate, fumarate, tartrate, citrate and lactate; examples of metal salts are alkali metal salts such as lithium salt, sodium salt, potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt; examples of ammonium salts are the ammonium salt and the tetramethylammonium salt; examples of organic amine additive salts are salts with morpholine and piperidine; and examples of amino acid additive salts are salts with glycine, phenylalanine, glutamic acid and lysine.

The compounds prepared herein, including those determined by the present methods, may be formulated into pharmaceutical compositions by mixing with pharmaceutically acceptable non-toxic excipients and carriers. Such compositions may be formulated for use in parenteral administration, especially in the form of liquid solutions or suspensions; or oral administration, especially in the form of tablets or capsules; or intranasally, especially in the form of powders, nasal drops or aerosols; or skin, for example by transdermal patches.

The composition may be conventionally administered in a single dose and may be prepared by any of the methods well known to those skilled in the art, for example, as described in Remington's

Pharmaceutical Sciences (Mack Pub.Co., Easton, PA, 1980). Mixtures for parenteral administration may include, as usual fillers, sterile water or physiological serum, polyalkylene glycols such as polyethylene glycol, terrestrial and vegetable oils, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer or polyoxyethylene-polyoxypropylene copolymers can be useful fillers to control the release of the active compounds. Other potential useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain as fillers, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate or fat solutions for administration in the form of nasal drops or as an intranasal gel. Compositions for parenteral administration may include glycocholate for buccal administration, salicylate for rectal administration, or citric acid for vaginal administration. Preferred compositions for transdermal patches are lipophilic emulsions.

The compounds of this invention can be used as a single active substance in a pharmaceutical composition. Alternatively, they may be used in combination with other active ingredients, for example, other growth factors that facilitate neural survival or axonal regeneration in diseases or disorders.

The compounds of the invention and the pharmaceutically acceptable derivative salts may be administered orally or orally, for example, as an ointment or injection. The concentrations of the compounds of this invention in the therapeutic mixture may vary. The concentration will depend on factors such as the total dosage of the drug to be administered, the chemical characteristic (e.g.,

hydrophobicity) of the administered compounds, route of administration, age, body weight and patient symptoms, etc. The compounds of this invention are typically prepared in aqueous saline containing about 0.1 to 10% weight / volume of parenteral compound. The typical dose ranges from about lpg / kg to about lg / kg body weight per day; preferred ranges are from about 0.01 mg / kg to 100 mg / kg body weight per day and preferably about 0.1 to 20 mg / kg one to four times daily. The preferred dosage of the drug to be administered depends on various things, such as the type and degree of progression of the disease or disorder, the overall health of the patient, the relative biological efficacy of the compound selected and the composition of the compound of the compound and method of administration .

The compounds of the invention, including the test compound and the compounds determined by the methods of the present invention and the pharmaceutically acceptable derivative salts, may be administered alone or in the form of various pharmaceutical compositions according to the pharmaceutical action and the purpose of the application. The pharmaceutical compositions according to the present invention can be prepared by equally mixing an effective amount of a compound or derivative of a pharmaceutically acceptable salt as the active ingredient with a pharmaceutically acceptable carrier. The carrier may be of various forms according to the form of the composition suitable for administration. If desired, such pharmaceutical compositions are formulated as a single dose suitable for oral or oral administration. Oral administration forms include ointment and injection.

The tablets may be prepared using excipients such as lactose, glucose, sucrose, mannitol and methyl cellulose, disintegrants such as starch, sodium alginate, calcium carboxymethyl cellulose and crystalline cellulose, lubricants such as magnesium stearate, tartaric acid, stearate, substances such as magnesium stearate, such as gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxypropyl cellulose and methyl cellulose, surfactants such as sucrose fatty acid ester and sorbitol fatty acid ester and the like the tea way. Preferably, each tablet contains 15-300 mg of the active ingredient.

The granules can be prepared using fillers such as lactose and sucrose, disintegrators such as starch, binders such as gelatin and the like in the conventional manner. The powders may be prepared using fillers such as lactose and mannitol and the like in the conventional manner. The capsules can be prepared using gelatin, water, sucrose, gum arabic, sorbitol, glycerin, crystalline cellulose, magnesium stearate, talc and the like in the usual way. Preferably, each capsule contains 15-300 mg of the active ingredient.

Syrup formulations can be prepared using sugars such as sucrose, water, ethanol and the like in the usual way.

Ointments can be prepared using ointment bases such as petroleum jelly, liquid paraffin, lanolin and macrogol, emulsifiers such as sodium lauryl lactate, benzalkonium chloride, sorbitan mono fatty acid ester, sodium carboxymethyl cellulose and gubiarabic.

Injectable preparations can be prepared using solvents such as water, saline, vegetable oils (eg olive oil and peanut oil), ethyl oleate and propylene glycol, solubilizers such as sodium benzoate, sodium salicylate and urethane, isotonic substances, such such as sodium chloride and glucose, preservatives such as phenol, cresol, p-hydroxybenzoate ester and chlorobutanol, antioxidants such as ascorbic acid and sodium pyrosulphite and the like in the conventional manner.

The invention is further illustrated by the following examples which are intended to elucidate the invention. These examples are neither intended nor interpreted as limiting the scope of the foregoing.

EXAMPLES

Example 1: General description of synthesis processes and examples

The general synthesis method used for the preparation of the bridged indenopyrrolcarbazoles of this invention of Formula II is shown in Figures 1 and 2. The general procedures for the synthesis of indenopyrrolocarbazole (III) / (VHI) can be performed as described in US Patent No. 4,859,256. No. 5,705,511, the disclosure of which is incorporated herein by reference in its entirety. When R ¹ is H, the lactam nitrogen of indenopyrrolcarbazoles (III) / (VIII) is protected by a suitable protecting group leading to (IV) / (IX). The protected compounds are treated with a suitable base in anhydrous organic solvent (s), which results in the formation of a dark red solution known to be a carbanion. Reaction of the carbanion with a bifunctional reagent (V) leads to an electrophilic attachment to the C = Y bond of (V) leading to the initial intermediate (VI) / (X). Treatment of intermediate (s) (VI) (X) and / or (VII) / (XI) with either sulfonic acid or with Lewis acid, for example boron trifluoro ether, yields bridged indenopyrrocarbazoles (1) / (11).

The lactam nitrogen protection strategy (shown in Figures 3 and 4) can be carried out by an acidic or substantially catalyzed process. The acid-catalyzed reaction can be carried out either with a synthetic resin substance as a binder allowing also the mobilization of indenopyrrocarbazole (1P) / (UP1) to a polymeric carrier, such as a polystyrene base, Rink acid resin (XI) (Figure 3) to give (XIII). Alternatively, the acid-catalyzed reaction may be carried out with a soluble substance

Preparation of compound (XIU) (Figure 4). The silyl protected compound (XV) is obtained by basic catalysis (Figure 4).

Figure 5 describes several methods for preparing intermediate (V). Procedure (a) describes the conversion of various acetals (XVI) to (XVII, 7 = bond). For example, ester-acetal / ketal (XVI, D = COOR) is completely reduced to the corresponding alcohol and subsequently oxidized (e.g., Swem or Dess-Martin oxidation) to aldehydacetal / ketal (XVII, K ⁸ = H). Alternatively, ester-acetal / ketal (XVI, D = COOR) is partially reduced with DIBAL to directly afford aldehyde (XVII, R ⁸ = H). Similarly, reduction of nitrile-acetal (XVI, D = CN) with DIBAL gives aldehyde (XVII, R ⁸ = H). Ketoacetals / ketal were prepared by attaching Gridnard reagents to Weinreb amide-acetal / ketal (XVI, D = CON (OMe) Me).

An intermediate (XVII, 7 ^ bond) can also be obtained by the two-step procedure outlined in Procedure (b). The addition of an organometallic reagent (XIX) to acetal / ketal (XVIII) yields alkene (XX) which, upon ozonolysis, followed by reductive treatment, yields ketoacetal / ketal (XVII). The preparation of the intermediate (XVII, Z = -x ether atom) by a two-step procedure is outlined in Procedure (c). Acetal (XXP) bonding with alkene (XXI) followed by ozonolysis (reductive treatment) of the resulting alkene yields ketoacetal / ketal (XVII). Alternatively, the intermediate (XVII, / = heteroatom) may be prepared by the two-step procedure outlined in Procedure (d). Reaction of compound (XXIV) with acetal / ketal (XVHI) yields (XXV), which is converted to keto-acetal / ketal (XVII) by the methods described in Procedure (a). Condensation of ketoacetal / ketal (XVII) with hydroxylamines, hydrazines, X-alkyl-alkoxyamines and amines yields an intermediate (XXVI) having electrophilic C = N functionality.

Indenopropylcarbazole with synthetic resin as binder (X1P) (Figure 6, Method A) is treated with excess Grignard

reagent as a base, which results in the formation of a dark red solution of carbanion. Subsequent reaction with (V) results in products resulting from electrophilic addition to the C = Y group. Aqueous treatment and cleavage of the product (s) with dilute acid (1% TFA in methylene chloride) from the resin results in the isolation of compound (s) (XXVII) and / or (XXVIII). Treatment of intermediate (s) (XXVII) and / or (XXVIII) with either sulfonic acid or Lewis acid, e.g. boron trifluoride etherate gives bridged indenopyrrolcarbazoles (II).

A similar strategy is used for the reaction of the lactam soluble protected intermediate, for example (XU) (Figure 7, Method B). In this case, however, the intermediate (XV) was treated with Triton B in pyridine as the base instead of the Grignard reagent. The intermediate (s) (XXIX) and / or (XXX) can be isolated with an intact lactam protecting group that can be chromatographically purified. As in method A (Figure 6), treatment with Lewis acid (such as boron trifluoride etherate) yields bridged indenopyrrocarbazoles (II), where R ^{1 =} H.

The introduction of groups R ³ , R ⁴ , R ⁵ and R ⁶ can be carried out as described in US Patent Nos. 5,705,511 and 4,923,986, the disclosure of which is incorporated herein by reference in its entirety. The R ³ substituent may be otherwise introduced after the formation of bridged indenopyrrolcarbazoles, as shown in Figure 8. Position 3 of the B ring is brominated with NBS to give compound (XXXI). A carbon moiety was then introduced using palladium-catalyzed reactions of Stille, Suzuki, Heck, Kumada or Castro-Stephens to obtain compounds of type (XXX), (XXX) and the like. In addition, compound (XXXI) can provide access to compounds where the bromine group is substituted with a heteroatom, for example, an amino base group using palladium catalyzed aminization of Buchwald.

Through the oxidation process, the oxygen-bound group can be introduced to the indene carbon of the E ring, as shown in Figure 9, compound (XXXTV). This chemistry of the process also leads to oxidation of the methylene group of the lactam (A ring), yielding an imide derivative, as shown below.

Example 2: Preparation of Rink Resin Intermediates: (XIII-A), (ΧΙΠ-B) and (ΧΙΠ-C), (Figure 3) Example 2-A

A three-necked round-bottom flask coupled to a suspended mechanical stirrer and a Dean-Stark separator were sequentially charged with Rink acid resin XII (10.00 g, 0.64 mmol / g), 1-methyl-2-pyrrolidinone (80 mL), benzene (350 mL), VIII -A (A ^ A ^ H 2, B ', B ² = O, R ³ = R ⁴ = R ⁵ = R ⁶ = H)) (3.00 g) and p-toluenesulfonic acid (1.00 g). The reaction mixture was refluxed for 20 hours and then filtered. The resin was washed with THF (5x175 mL) and the filtrate was set aside. The resin was then washed sequentially with DMSO (4x100 mL), 2% aqueous NaHCO ₃ (4x100 mL), water (4x100 mL), DMSO (2x200 mL), THF (4x100 mL) and ethyl acetate (4x100 mL). The resin was dried under vacuum (24 hours) to give 11.70 (0.47 mmol / g) of the binder resin VJll-A, (XIII-A).

The primary THF washings were evaporated, the residue was diluted with water (750 mL) and the resulting precipitate filtered off and washed successively with water, 2% aqueous NaHCO ₃ (4x100 mL) and water (4x100 mL). After drying under vacuum, VIII-A (1.28 g) was found.

Example 2-B

Similarly, VIII-B (Α ', Α ^ Ο, B ^ B ^ H 2, R ³ = R ^4; = R ^5: = R ⁶ = H), (0.5 g) was bound to Rink acid resin XP ( 1.52 g) to obtain 1.58 g of the VIII-B binder resin (ΧΙΠ-Β).

Example 2-C

Similarly, VIII-C (A ¹ , A ² = H ₂ , B ¹ , B ² = O, R ³ = R ⁴ = R ⁵ = H, R ⁶ = 10OMe) (1.02 g) was coupled to the Rink acid resin XP (3.12 g) to give 3.70 (0.46 mmol / g) from the compound of the binding resin VIII-C, (XIII-C), together with the recovered compound XP1-C (0.44 g).

Example 3: Preparation of Compound (P-1), Compound (P-2), Compound (P-3), Compound (P-4a), Compound (P-4b), Compound (II-6) and Compound (P -8) (Method A, Figure 6) Example 3-A

To a suspension of (XIII-A) (1.25 g) in THF (24 mL) was added a 1.0 M solution of EtMgBr (6.25 mL in THF) and the reaction mixture was stirred for 1 hour before the addition of NMPA (5.0 mL). ). After stirring for 10 minutes, diethoxybutyraldehyde (3.0 g) (which was prepared according to the procedure described in the literature of Paquette, et al., J. Am.Chem.Soc., 1997, 119, 9662-71) was added and the reaction mixture was stirred. for 20 hours. The reaction mixture was cooled sharply with 10% aqueous NH4Cl (5 mL) and filtered. The resin was washed successively with 10% aqueous NH4Cl (3x10 mL), water (3x10 mL), THF (3x10 mL), DMF (3x10 mL), water (3x10 mL), THF (3x10 mL) and ether (3x10 mL). The resin was dried in vacuo, dissolved in methylene chloride (15 mL) and treated with trifluoroacetic acid (0.15 mL). After stirring for 1 hour, the reaction mixture was filtered and the filtrate was evaporated. The resulting residue was dissolved in methylene chloride (20 mL) and treated with pyridinium tosylate (50 mg) and the resulting solution stirred for 4 hours. At this time, the reaction mixture was washed with saturated aqueous NaHCO ₃ and brine and dried over MgSO ₄ .

After filtration and evaporation of the solvent, the residue was purified by preparative HPLC (Zorbax RX-8, 4 × 25 cm, washed with

60% MeCN / water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO ₃ and extracted with metal chloride (3 x 50 mL) and dried over MgSO ₄ . Filtration and evaporation of the solvent gave 70.2 mg of compound II-1 as a white powder having the following characteristic: ¹³ C NMR (DMSO-de) δ 171.8, 143.3,

142.4, 141.4, 140.1, 140.0, 136.6, 129.2, 127.9, 127.4, 127.1, 126.8, 124.1 (2C), 122.7, 121.6, 121.5, 118.3, 112.1, 88.1, 79.2, 56.6, 45.6, 33.4, 24.8; ^! H NMR (DMSO-d6) d 9.21 (d, 7 = 7.5, 1H), 8.62 (s, 1H), 7.98 (d, J = 7.7, 1H), 7.86 (d, 7 = 8.3, 1H). 7. 71 (d, J = 7.3, 1H), 7.49 (dd, 7 = 7.9, 7.4, 1H), 7.41 (dd, J = 7.5, 7.4, 1H), 7.36 - 7.27 (m, 2H). 6.86 (d, 7 = 6.0, 1H), 5.63 - 5.58 (m, 1H), 4.91 (s, 2H), 4.53 (d, 7 = 3.3, 1H), 2.23 - 2.14 (m, 1H), 1.96 - 1.92 (m, 1H), 0.96-0.88 (m, 1H), 0.60-0.57 (m, 1H); MS m / z (M + H) calcd 379, found 379.

Also, by preparative HPLC of this reaction mixture of the product, compound P-2 (0.5 mg) was isolated which had the following characteristic ^:! H NMR (DMSO-d6) δ 9.17 (d, 7 = 8.1, 1H), 8.62 (s, 1H), 7.98 (d, 7 = 7.0, 1H), 7.85 (d, 7 = 6.8, 1H), 7.57 (d, 7 = 6.8, 1H), 7.49 (dd, J = 7.9,

7.4, 1H), 7.44 - 7.26 (m, 3H), 6.81 (d, 7 = 6.0, 1H), 5.43 - 5.33 (m, 1H), 4.43 (s, 2H), 2.23 - 2.14 (m, 1H) , 1.96-1.92 (m, 1H), 1.45-1.55 (m, 2H), 0.960.88 (m, 1H), 0.60-0.57 (m, 1H), 0.29 (t, 7 = 7.0, 3H); MS m / z (M + H) calcd 407, found 407.

Example C-B

Similarly, as described above for compound II-1, resin (XIII-A) (70.3mg) was treated with 1,1-diethoxy-2-pentanone (0.75 mL) (which was prepared according to the known from procedure of Sworin.c ⁷ / al., J. Org. CAem., 1988,53,4894-6) to obtain compound II-3 (3.5 mg), which was isolated by preparative TLC (silica gel eluted with 50% EtOAc / toluene) and has the following characteristics ^:! H NMR (DMSO-d6) δ 9.42 (d, J = 8.2, 1H), 8.58 (s, 1H), 7.95 (d, J = ΊΑ, 1H), 7.79 (d.

J = 8.3, 1H), 7.71 (d, J = 7.1), 7.50 - 7.20 (m, 4H), 6.81 (d, J = 5.9, 1H), 4.90 (s, 2H), 4.46 (s, 1H). 2.35-2.20 (m, 1H), 1.98 (s, 3H), 1.75 - 1.60 (m, 1H),

1.25 - 1.00 (m, 1H), 0.35 - 0.15 (m, 1H); MS m / z (M + H) calcd 393, found 393.

Example 3-C

Similarly, (XIII-A) (74.3 mg) was treated with 1,1-diethoxy-2hexanone (which was prepared according to the well-known Brenner procedure, J. Org. Chem., 1961,16,22-7). (0.75 mL) to obtain compound P-4a, (2.10 mg) and compound P4b (1.06 mg), which were isolated individually by preparative HPLC (Zorbax RX-8, 4 x 25 cm, 65% MeCN / water w / 0.1 % trifluoroacetic acid). Compound II-4a has the following characteristics: 1 H NMR (DMSO-d ₆ ) δ 9.30 (d, J = 8.3, 1H), 8.55 (s, 1H), 7.97 (d, ./= 7.2, 1H), 7.65 ( d, J = 8.5, 1H), 7.59 (d, J = 7.5,), 7.48 (dd, J = 7.8, 7.2, 1H) 7.39 - 7.15 (m, 3H), 6.31 (dd 5.9, 5.5, 1H). 5.02 (s, 1H), 4.88 (s, 2H), 0.88 (s, 3H) other aliphatic signals were lost below the peaks of the solvent; MS m / z (M + H) calcd 407, found 407. Compound II-4b has the following characteristics: 1 H NMR (DMSO-d) d 9.43 (d, J = 8.1, 1H), 8.59 (s, 1H). 7.99 (d, J = 7.3, 1H), 7.75 - 7.65 (m, 2H), 7.49 (dd, J = 7.0, 6.4, 1H), 7.43 (dd, J = 8.2, 8.1, 1H), 7.36 - 7.25 ( m, 2H), 6.75 (s, 1H), 4.91 (s, 2H), 4.50 (s, 1H), 1.95 (s, 3H) other aliphatic signals are lost below the peaks of the solvent; MS m / z (M + H) calcd 407, found 407.

Example 3-D

Similarly, (XIII-C) (1.00 g) was treated with diethoxybutyraldehyde (3.65 g) to give compound P-6 (87.8 mg), which was isolated by preparative HPLC (Zorbax RX-8, 2.5 x 25 cm, 65% MeCN / water w / 0.1% trifluoroacetic acid) and has the following characteristics: 1 H NMR (DMSO-d ₍₎ ) δ 9.09 (d, J- 8.6, 1H), 8.60 (s, 1H),

7.95 (d, J = ΊΑ, 1H), 7.84 (d, J = 8.3, 1H), 7.47 (dd, J = 7.2, 7.0, 1H), 7.35 (s.

1H), 7.29 (dd, 7.0, 7.0, 1H), 6.98 (dd, J = 8.6, 1.9, 1H), 6.83 (d, J = 6.0,

1H), 5.65-5.55 (m, 1H), 4.88 (s, 2H), 4.48 (d, J = 3.9, 1H), 3.82 (s, 3H), 2.25

- 2.10 (m, 1H), 2.08 - 1.85 (m, 1H), 0.96 - 0.75 (m, 1H), 0.65 - 0.50 (m, 1H);

MS m / z (M + Na) calcd 431, found 431.

Example C-E

Similarly, resin (XIII-B) (153.2 mg) was treated with diethoxybutyraldehyde (1.5 mL) to obtain compound P-8 (3.6 mg), which was isolated by preparative HPLC (Zorbax RX-8, 2.5 x 25 cm) , 65% MeCN / water w / 0.1% trifluoroacetic acid) and has the following characteristics: 1 H NMR (DMSO-c 3) δ 9.09 (d, <7 = 7.9, 1H), 8.81 (s, 1H), 7.81 - 7.73 (m , 3H), 7.48 - 7.35 (m, 3H), 7.24 (dd, J = Ί.6, 7.5, 1H), 6.85 (d, J = 6.2, 1H), 5.63 - 5.59 (m, 1H), 4.86 ( s, 2H), 4.61 (d, J = 3.6, 1H), 3.82 (s, 3H), 2.21 - 2.13 (m, 1H), 1.96 - 1.90 (m, 1H), 0.87 - 0.79 (m, 1H). 0.61 0.56 (m, 1H); MS m / z (M + H) calcd 379, found 379.

Example 4: Preparation of Compound P-7a and Compound P-7b (Method A, Figure 6)

Example 4-A

Preparation of (1,1-diethoxyethoxy) acetone

To a cold (0 ° C) suspension of NaH (2.68 g, 60%) in THF (150 mL) was added to a solution of 1,1-diethoxyethanol (which was prepared according to the procedure known in the literature by Zirkle, et al., J. Org. Chem., 1961, 26, 395-407) (9.00 g) in THF (20 mL) and the reaction mixture was stirred at room temperature for 1 hour before the addition of methyl chloride (8.0 mL). The reaction mixture was refluxed overnight, cooled and filtered through a pad of villages. The solvent was removed by rotary evaporation and the residue was purified by column chromatography (silica, 20% ether / hexane) to give 1,1-diethoxyethyl methyl ether (11.5, 90%). Ozonolysis of a cooled (-30 ° C) solution of this ether (6.00 g) in EtOAc (80 mL) was carried out until starting material was established by TLC (1 hour). At this time, the reaction mixture was purged with oxygen, treated with Pd (OH) ₂ (150 mg) and stirred under hydrogen overnight. The catalyst was filtered off and the filtrate was concentrated by rotary evaporation. The resulting residue was purified by column chromatography (silica, 20% EtOAc / hexane) to give the title compound (4.53 g, 82%).

Example 4-B

According to Method A (Figure 6), the resin (XIII-A) (230.2 mg) was treated with EtMgBr (1.25 mL) followed by (1,1-diethoxyethoxy) acetone (Example 3-A) (1.2 mL). After treatment and removal from the resin, part of the crude reaction mixture of the product (10.5 mg) was dissolved in metal chloride (20 mL) and treated with BF ₃ etherate (20 µL). After stirring for 2.5 hours, the solution was washed with saturated aqueous NaHCO ₃ and brine before drying over MgSO ₄ ,. After filtration and removal of the solvent, the resulting residue was purified by preparative HPLC (Zorbax RX-8, 4 x 25 cm, 65% MeCN / water w / 0.1% trifluoroacetic acid) to give compound P-7a (2.34 mg) and compound P-7b (1.34 mg). Compound (II-7a) has the following characteristics: * H NMR (CDCl ₃ ) δ 9.35 - 9.20 (m, 1H), 7.87 (d, J = 7.6, 1H), 7.62 (d, J = 7.0, 1H), 7.60 - 7.45 (m, 1H), 7.49 (dd, J = Ί.Ί, 7.5, 1H), 7.40 (d, J = 8.1, 1H), 7.37 - 7.26 (m, 3H), 6.22 (s, 1H). 5.20 - 4.85 (m, 1H), 4.47 (s, 1H), 3.67 (d, J = 12.7, 1H) 3.52 (d, J = 11.8, 1H), 3.40 (d, J = 12.7, 1H), 3.38 ( d, J = 11.8, 1H), 1.91 (s, 3H); MS m / z (M + H) calc'd 409, found 409. Compound II-7b has the following characteristics: * H NMR (CDCl ₃ ) δ 9.58 - 9.22 (m, 1H), 7.82 (d, 7.4, 1H), 7.60 -7.40 (m, 3H), 7.37 - 7.27 (m,

3H), 7.21 (d, J = 8.1, 1H), 5.81 (s, 1H), 5.21 (s, 1H), 5.10 - 4.80 (m, 1H), 4.59 (d, J = 13.5, 1H), 4.38 ( dd, J = 13.5, 5.3, 1H), 4.21 (d, 13.1, 1H), 3.82 (d,

J = 13.2, 1H), 1.13 (s, 3H); MS m / z (M + H) calcd 409, found 409.

Example 5: Preparation of Compound P-5 (Figure 8)

To a solution of compound II-1 (8.1 mg) in THF (2 mL) was added NBS (4.6 mg) and the reaction mixture was stirred overnight. More NBS (4.5 mg) was added and the reaction mixture was stirred for 2.5 hours. The insoluble material was filtered off and the filtrate was concentrated by rotary evaporation. The resulting residue was purified by column chromatography (C-18, 65% MeCN / water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO ₃ and extracted with methylene chloride (3 x 20 mL) and dried over MgSO 4. After filtration and evaporation of the solvent, compound P-5 (5.1 mg) was obtained as a white powder having the following characteristic: Ή NMR (DMSO-cC) δ 9.22 (d, J = 7.4, 1 Η), 8.67 (s, 1Η), 8.14 (s, 1H), 7.86 (d, J = 8.7, 1H), 7.72 (d, J = 7.0, 1H), 7.63 (d, J = 7.8, 1H), 7.42 (dd, J = 7.5, 7.3, 1H), 7.35 (dd, J = 7.3, 7.2, 1H), 6.86 (d, J = 6.0, 1H), 5.63-5.58 (m, 1H), 4.94 (s, 2H), 4.54 (d , J = 3.1, 1H), 2.30 - 2.14 (m, 1H), 2.00 - 1.82 (m, 1H), 0.96 - 0.88 (m, 1H), 0.62 - 0.50 (m, 1H); MS m / z (M + H) calcd 457/9 (1: 1) found 457/9 (1: 1).

Example 6: Preparation of Intermediate XV (Figure 4)

To a solution of VHI-A [ΑζΑΜίζ, B ^ B ^ O, R ³ = R ⁴ = R ⁵ = R ⁶ = H) J (1.05g) in DMF (25 mL) was added triethylamine (0.75 mL) and t -butyldimethylsilyl chloride (TBS-C1) (0.65 g). After stirring for 3 hours, the reaction mixture was quenched with saturated aqueous NaHCO ₃ and extracted with EtOAc. The organic layer was washed with water and brine and dried over MgSO ₄ . After filtration and evaporation of the solvent, the resulting residue was triturated with ether to give compound XV (848 mg). The washings were evaporated to leave a residue which was purified by column chromatography (silica, 1%

EtOAc / CH ₂ C12) gave an additional product (502 mg, combined yield of 94%) having the following spectral characteristics: * H NMR (DMSOf) δ 11.94 (s, 1H), 9.32 (d, J = 7.6, 1H ), 8.03 (d, J = 7.7, 1H), 7.64 (d, J = 12, 1H), 7.58 (d, J = 8.1, 1H), 7.44 (dd, J = 7.7, 7.6, 1H), 7.39 ( dd, J = 7.7, 7.6, 1H), 7.32 (d, J = 7.3, 1H), 7.25 (dd, J - 7.6, 7.3, 1H), 5.00 (s, 2H), 4.14 (s, 2H), 0.99 (s, 9H), 0.46 (s, 6H); MS m / z (M + H) calcd 425, found 425.

Example 7: Preparation of Compound P-1 by Method B (Figure 7)

A solution of Triton B in pyridine (0.45 M) was prepared by diluting a 40% solution of Triton B in acetanol (10 mL) in pyridine (10 mL). The solvent was removed under reduced pressure (20 mm Hg) to a final volume of ~ 8 mL. The residue was diluted with pyridine to 50 mL, filtered and stored under nitrogen. A solution of XV (20.3 mg) in pyridine (2.0 mL) was purged with argon and treated with 300 µL of Triton B (0.45 M in pyridine) and diethoxybutyraldehyde (50 µL). After stirring for 2 hours, the reaction mixture was extracted into EtOAc, washed with 1N aqueous HCl, aqueous brine and dried over MgSO ₄ . After filtration and evaporation of the solvent, the coupling product was dissolved in CH ₂ Cl ₂ (10 mL) and treated with BF ₃ etherate (10 pL). After stirring for 2.0 h, the solution was washed with saturated aqueous NaHCO ₃ and brine before drying over MgSO ₄ . Removal of the solvent by rotary evaporation gave a residue which was purified by preparative HPLC (Zorbax RX-8, 2.5 x 25 cm, 65% MeCN / water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO ₃ and extracted with methylene chloride (3 x 20 mL) and dried over MgSO ₄ . Filtration and evaporation of the solvent gave N-1 (11.8 mg, 65% yield) whose 1 H NMR and MS spectrum and HPLC retention time were identical to those of the prepared and isolated substance by Method A described in Example 3 -A.

Example 8: Preparation of Compound P-9 (Figure 8)

To a suspension of bromo compound N-5 (6.2 mg) in 1-propanol (4.0 mL) was added 3-aminophenylboronic acid (3.8 mg). After stirring for 0.25 h, Pd (OAc) ₂ (2.0 mg) Ph ₃ P (4.8 mg), Na ₂ CO ₃ (2.8 mg) and water (2.0 ML) were added sequentially. The mixture was refluxed for 0.75 hours, cooled, extracted into CH ₂ Cl ₂ and washed with water and brine. The organic layer was dried over MgSO4 and the solvent was removed by rotary evaporation to obtain a residue which was purified by preparative HPLC (Zorbax RX-8, 2.5 χ 25 cm, 50% MeCN / water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO ₃ and extracted with methylene chloride (3 x 20 mL) and dried over MgSO 3. Filtration and evaporation of the solvent gave compound P-9 (3.1 mg, 49% yield) and had the following spectral characteristics: ¹ H NMR (DMSO-d ₆ ) δ 9.22 (d, J = 7.5, 1H), 8.66 ( s, 1H), 8.00 - 7.25 (m, 8H), 7.12 (dd, J = 7.1, 7.0, 1H), 6.95 - 6.80 (m, 3H), 6.53 (d, J = 6.0, 1H), 5.63 - 5.58 (m, 1H), 4.99 (s, 2H), 4.55 (s, 1H), 2.25-2.10 (m, 1H), 1.95 -1.90 (m, 1H), 0.98-0.88 (m, 1H), 0.65-0.57 (m, 1H); MS m / z (M + H) calcd 470, found 470.

Example 9: Preparation of Compound 11-10 (Figure 9)

To a solution of compound II-1 (5.0 mg) in DMSO (1 mL) was added NaCN (4.3 mg) and the mixture heated to 145 ° C for 1 hour. The mixture was cooled in EtOAc and washed with water (3 x 20 mL) and brine. The organic layer was dried over MgSO4, filtered and evaporated to give a residue which was purified by preparative HPLC (Zorbax RX-8, 2.5 χ 25 cm, 55% MeCN / water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO ₃ , extracted with methylene chloride (3 x 20 mL) and dried over MgSO ₄ . Filtration and evaporation of the solvent gave compound ΠΙΟ (2.7 mg, 50% yield) and had the following spectral characteristics: 1 H

NMR (DMSO-d6) δ 11.4 (s, 1H), 8.86 (d, J = 7.9, 1H), 8.79 (d, J = 7.6, 1H),

7.90 (d, ./8.3, 1H), 7.62 - 7.55 (m, 2H), 7.49 (dd, J = 7.6, 7.4, 3H), 7.40 (dd,

J = ΊΑ, 7.3, 1H), 7.35 (dd, 7.5, 7.4, 1H), 6.86 (d, J = 6.O, 1H), 6.03 (s, 1H),

5.40 - 5.30 (m, 1H), 2.25 - 2.14 (m, 1H), 2.03 - 1.90 (m, 1H), 1.10 - 0.98 (m,

1H), 0.82-0.77 (m, 1H).

Example 10: Preparation of Compound P-11 (Method A, Figure 6)

According to Method A, the resin (XIIIa) (150.2 mg) was reacted with EtMgBr (1.0 mL) followed by ethyl 2,5-dioxopentanoate (Schmidt, er al., Synthesis, 1993, 809) (1.5 mL). After treatment and cleavage of the resin, the crude product reaction mixture was dissolved in methylene chloride (20 mL) and treated with BF ₃ etherate (20 mL). After stirring for 2.5 hours, the solution was washed with saturated aqueous NaHCO ₃ and brine before drying over MgSO ₄ . After filtration and removal of the solvent, the resulting residue was purified by preparative HPLC (Zorbax RX-8, 4 x 25 cm, 55% -75% gradient MeCN / water w / 0.1% trifluoroacetic acid) to give compound II-11 (6.4 mg), which has the following characteristics: '11 NMR (DMSO-cC) δ 9.36 (d, J = Ί.Ί, 1H), 8.68 (s, 1H), 8.00 (d, J = 7.7, 1H), 7.83 ( d, J = 8.3, 1H), 7.58-7.15 (m, 5H), 6.97 (d, J = 5.9, 1H), 4.93 (s, 2H), 4.82 (s, 1H), 4.48 (q, J = 7.1) , 2H), 2.42 - 1.91 (m, 2H), 1.37 • (t, 3H, J = 7.1), 1.25 - 0.63 (m, 2H).

Example 11: Preparation of Compound P-12

A solution of compound N-11 (3.4 mg) in THF (2 mL) was treated with a 2 M solution of LiBH ₄ (1.0 mL in THF) and the reaction mixture was stirred for 1.5 h. The reaction mixture was cooled sharply by the addition of 1 N aqueous HCl (4 mL). After stirring for 20 min, 10% aqueous NaOH (15 mL) was added and the mixture was extracted with methylene chloride (3 x 10 mL). After drying over MgSO ₄ , the mixture was filtered and the solvent was evaporated to give compound P-12 (0.32 mg), which had the following characteristics: 1 H NMR (DMSO-c 3) δ 9.35 (d, J = Ί.Ί, 1H) , 8.62 (s, 1H), 7.98 (d, J = Ί.Ί, 1H), 7.83 (d, J = 8.2, 1H), 7.75 (d, J = 8.2, 1H), 7.50 - 7.25 (m, 4H) ), 6.84 (d, J = Ί.Ί, 1H), 6.11 (s, 1H), 4.91 (s, 2H), 4.71 (s, 1H), 4.50 - 4.40 (m, 1H), 4.30 - 4.20 (m , 1H), 2.42 - 1.91 (m,

2H), 1.25 - 0.63 (m, 2H); MS m / z (M + H) calcd 409, found 409.

Example 12: Enhanced Spinal Cord ChAT Activity

ChAT is a specific biochemical marker for functional cholinergic neurons. Cholinergic neurons represent the main cholinergic access to the hippocampus, olfactory nuclei, u interpeduncular nuclei, cortex, cerebral tonsils, and parts of the thalamus. In the spinal cord, motoneurons are cholinergic neurons that contain ChAT (Phelps, et al., J. Comp. Neurol., 1988, 273, 459-472). ChAT activity is used to study the effects of neurotropins (e.g., NGF or NT-3) on the survival and / or function of cholinergic neurons. The ChAT study also serves as an indicator of the regulation of ChAT levels in cholinergic neurons.

Methods-. Rat fetal spinal cord cells were dissociated and experiments were performed as described (Smith, et al., J. ® Cell Biology, 1985, 101, 1608-1621; Glicksman, et al., J. Neurochem., 1993,

61, 210-221). Dissociated cells were prepared from spinal cord dissected from rats (embryonic age 14-15 days) by standard trypsin dissociation techniques (Smith et al., Supra.). Cells were placed in wells coated with poly-1-ornithine dermal tissue culture at 6 x 10 ³ cells / cm ² in serum-free N2 medium supplemented with 0.05% bovine serum albumin (BSA) (Bottenstein, et al., Proc. Natl. Acad Sci. USA, 1979, 76, 514-517). The cultures were incubated at 37 ° C in humidified atmospheric air at 5% CO ₂ /95% for 48 hours. ChAT activity was measured after 2 days in vitro using a modification of the Fonnum procedure (Fonnum, Neurochem., 1975, 24,

407-409) according to McManaman, et al. and Glicksman, et al. (McManaman, et al., Develop. Biol., 1988, 125, 311-320; Glicksman, et al., J. Neurochem., Supra.).

The compounds of formula II described in the examples are listed in

Table 2.

The values for R ¹ , R ⁴ , R ⁶ and R ⁷ are Η; Y is O; and η is 1.

Table 2

Compound No. AiA ₂ BjB ₂ r ₂ Ks r ₅ r ₈ Z m II-1 0 n, n n n Η Η connection 1 P-2 0 n, n Et n Η Η connection 1 P-S 0 n, n n n Η Me connection 1 P-4a 0 n, n n n Η Me connection 2 N-4H 0 n, n n n Η Me connection 2 P-5 0 n, n n Br Η Me connection 1 II-6 0 n, n n n 10- ΟΜ e Η connection 1 P-7a 0 n, n n n Η Me 0 1 P-7b Fr. n, n n n Η Me 0 1 P-8 n, n 0 n n Η H connection 1 P-9 0 n, n n 3- nh ₂ -Ph Η H connection 1 P-10 0 0 0 n Η Η H connection 1 II-11 0 n, n n Η Η CO ₂ -Et connection 1 P-12 0 n, n n Η Η CH ₂ -OH connection 1

Example 13: pCDNA3-EE-MLK3, pcDNA3-EE-MLK3 (K144R)

MLK was cloned as described (Lee, et al., Oncogene, 1993, 8, 3403-3410; Ezoe, et al., Oncogene, 1994, 9, 935-938). Cell DNA is prepared from 200 ng of polyadenylated melanocyte mRNAs and 5% of the reaction mixture is used as a template to extend the repertoire of RTK cell DNA using mixtures of either two or four highly degenerate oligonucleotide primary structures arising from the conserved sequence VI of IX known RTK subdomains: PTK1, 5'-CGGATCCACMGIGAYYT3 '(SEQ ID NO: 1); RTK2, 5′-GGAATTCCAWAGGACCASACRTC-3 '(SEQ ID NO: 2); RTKZ, 5'CGGATCCRTICAYMGIGAYYTIGCIGCIMGIAA-3 '(SEQ ID NO: 3); RTK4, 5'-GGAATTIAYIGGAWAIGWCCAIACRTCISW-3 '(SEQ ID NO: 4). Forty cycles of PCR were performed using Taq DNA polymerase (AmpliTaq; Perkin-Elmer / Cetus) and an automatic DNA thermal cycle; each cycle consists of 40 seconds at 94 ° C, 2 minutes at 37 ° C and 3 minutes at 63 ° C. The eight PCR products were combined, treated with DNA polymerase (Klenow), cleaved with BamH1 plus EcoR1 and electrophoresed on a 5% polyacrylamide gel. Ethidium bromide staining determined a predominant 200-230 bp band, which was excised, eluted and cloned into M13bp18. In one experiment, a portion of the PCR amplified by DNA was not cleaved, but nevertheless cloned bluntly into M13shp18 cleaved by Smal. The nucleotide sequence is determined by the chain interruption sequence method.

One cDNA, designated PTK1, was used as a probe for the study of human melanoma and melanocyte cDNA libraries. A clone designated RTK 1-3.2 includes the entire open reading frame 2541 nt, encoding a protein of 847 amino acids. This cDNA was excised with Ncol, blunted with DNA polymerase (Klenow), excised with EcoR1, and ligated into carrier pCDNA3-EE cut with BamH1, blunted and then cut with EcoR1. The pCDNA3-EE carrier was created by introducing into the HindIII / BamH1 region a primary codon encoding oligos, followed by an EE epitope, MEEEEYMPME (Sequence ID # 5) (Grussenmeyer, et al., Proc. Natl. Acad. Sci. USA 1985, 82, 7952-7954). The MLK3 dead kinase version was made by mutation of K144R using previously published PCR techniques (Chen, et al., Biotechniques, 1994, 17, 657-659). The first mutagenic oligo is 5′-GTGGCTGTGCGGGGCAGCTCGCCAG-3 ′ (Sequence ID # 6) and the second oligo is 5′-GAGACCCTGGATCTCGCGCTT-3 ′ (Sequence ID # 7). Using MLK3 as a provisional, these oligos are used in PCR to generate an 806 bp fragment and applied in the second PCR reaction using primary T7 as another enhancer and MLK3 as temporary to create a 1285 bp fragment. The fragment was separated by agarose gel electrophoresis, isolated, cloned into pGEM-5 (Promega) and the sequence of the amino acid residue lines in the proteins determined. The fragment was cut with HindIII and Hpal and introduced into pCDNA3-EEMLK3 excised with HindIII and Hpal. An additional mutation moment is determined at nucleotide 1342. To correct this, the PflM1 fragment (nt 1093-1418) is truncated by wild-type MLK3 and used to replace the identical fragment in the K144R mutated MLK3.

Example 14: pFB-FLAG-MLK3

To obtain the MLK3 protein, the cDNA was cloned into a rod-viral pFB-FLAG expression vector. MLK3 was excised from PTK13.2 by digestion with Ncol, thinned with DNA polymerase (Klenow), excised with Not1 and ligated into pFB-FLAG digested with Stul and Notl. From pFB (Life Technologies) is extracted pFB-FLAG, which has a coding sequence for the FLAG epitope (Hopp, et al., Biotechnology,

1988, 6, 1205-1210) with a primary codon, MDYKDDDDK (Sequ. ID no: 8) added to a poly-binding portion in the BamH1 region.

Example 15: pFB-GST-MLK3 (KD)

The rod-like viral expression of the kinase region of MLK3 was obtained by excision of the MLK3 fragment from pGEXKG-MLK3 (KD) using EcoR1 and Xhol and ligated into a pFB transmitter cut with EcoR1 and Xhol, where the GST encoding sequence (GST) cloned into a higher branch. This is achieved by obtaining the GST coding sequence and the polysampling portion of the pGEXKG carrier by PCR using the carrier as a template (Guan, et al., Anal. Bioch., 1991, 192, 262-267). The 5 'oligo for PCR creates a Bgl2 restriction site at the 5' end of the fragment. This isolated fragment was then digested with Bgl2 and HinD3 and ligated into pFB digested with BamHl and HinD3.

Example 16: pGEXKG-MLK3 (KD)

A fragment of cDNA that includes both the MLK3 kinase region and part of the leucine zigzag kinase (nt 736-1791) was obtained by PCR using PTK1 cDNA. The isolated fragment is digested by the restriction enzymes EcoR1 and Xho 1, sites that are included in the PCR oligo and cloned into pGEX-KG digested with EcoR1 and Xho 1. This fragment in pGEX-KG is then truncated by PCR, to to include only the kinase region (nt 736-1638).

Example 17: pKH3-MLK2, pKH3-MLK2 (KA)

MLK2 was cloned using degenerate PCR (Dorow, et al., Eur. J.

Biochem., 1993, 213, 701-710; Dorow, et al., Eur. J. Biochem., 1995, 234,

492-540). Segments of cDNA encoding protein kinase catalytic subunits expressed in the epithelial tumor cell line Colo are amplified by RNA by reversible transcriptase PCR. The degenerate PMH

primary PCRs are based on sequences encoding conserved motifs in subbands Vib and VIII of kinase catalytic regions of the epidermal growth factor receptor family. The sequences of the primers were as follows: early primer, 5'CGGATCCGTG (A) CACC (A) GT (CG) G (A) ACC (T) T-3 '(Sequence ID: 9), reversible primer, 5 '-GGAATTCACCA (G) TAA (G) CTCCAG (C) ACATC-3' (Sequence ID: 10). Several PCR products were cloned into M13 and in a specific sequence using the T7 Super-Base Sequence Detection Kit (Bresatec). A 216-bp PCR product was used as a sample to study the Xgtll human colon cDNA library (Clontech, catalog #HL 10346). The fragment was randomly labeled, hybridization was performed at 65 ° C, and the filters were washed to form a filament of 0.2X NaCl / Citrate (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.5% SDS at 65 ° C. The filters were authored for 16h at -70 ° C on a Kodak XAR-5 film. Four clones were isolated and the longest, 1.2 kb, was used to re-examine the same library using the same conditions. Four more clones are isolated and one of these clones represents a 1034 bp fragment of MLK2. This clone is used to study the human brain library XgtlO. Approximately 500,000 clones were assayed and one 3454 bp clone representing the entire coding region of MLK2 was isolated.

MLK2 is cloned, from ATG to polyA tail, into carrier pKN3 between BamH1 and EcoR1 sites in two steps, with a BamH1 site in the middle of the MLK2 sequence. The pKN3 carrier is formed by introducing three copies of the HA epitope outgrowth, followed by a BamH1 site between the Xbal and EcoR1 sites of the polysorbent pRK7. To make the mutagenized version, the K125A, MLK2 5 'BamHl fragment was cloned into the Promega pAlter carrier and mutated as recommended by i

manufacturer. The fragment was then cloned back into the MLK2 pKH3 carrier.

Example 18: pcDNA3-HA-JNKl

JNK1 cDNA was prepared as described (Coso, et al., Cell, 1995, 81, 1137-1146). cDNA was prepared by PCR using the human skeletal muscle (Invitrogen) cDNA template and cloned into Bgl2 / Sall sites of pcDNA3-HA, a modified pcDNA3 expression plasmid encoding the epitope (Wilson, et al., Cell, 1984, 37 , 767-778). This is then excised from pcDNA3, including the HA epitope and ligated into pGEX-4T3 (Pharmacia). The JNKI cDNA was excised from the construction of pGEX-4T3 as a Bgl2 / Sall fragment and ligated into pcDNA3-HA, a carrier with the HA epitope added to the HinD3 / BamH1 site of pcDNA3.

Example 19: pFLAG-DLK

DLK was cloned into the pcDNA3 expression carrier with the FLAG epitope added as described (Holzman, et al., J. Biol. Chem., 1994, 269, 30808-30817). A fragment of cDNA for DLK was isolated by degenerate PCR cloning based on an oligonucleotide. Total RNA was extracted from the kidney per embryo at 13.5 days (32 organs) and from the kidney per embryo at 17.5 days (16 organs) using the commercially available phenol / guanidine isothiocyanate reactive method according to the manufacturer's guidelines (TRIzol Reagent, Life Technologies, Inc.). Subsequent degradation with RNase-free DNase I, total RNA transcribes reversibly with RNase H-reversible transcriptase (Superscript, Life Technologies, Inc.) from oligo (cGG) synthetic primary oligonucleotide to single filamentous cDNA. The discharged primary oligonucleotides corresponding to the protein tyrosine kinase catalytic subunits Vib and IX, originally determined by Wilks (Wilks, Proc Natl. Acad Sci USA. 1989, 86, 1603-1607), were modified to 5 'EcoRI and Hind \\\ sites, respectively (567

ATAATTC (GT) GC (TAGC) GCCA (GA) GTC (TAGC) CGGTG-3 '(Sec.

ID: 11), 5'ATAAGCTTCC (TC) (AG) T (GC) AAGTGGA (TC) (GC) GC (AGC) CC (CT) GA3 ') (Sequence ID: 12). Forty PCR cycles were performed for 1.5 min at 94 ° C, for 2 min at 37 ° C, and for 3 min at 63 ° C. Fresh reagents were added and another 40 cycles were performed before the final 10-min extension at 72 ° C. The resulting 200-210-bp DNA expanded product was isolated in gel, subcloned into prepared pGEM7zf (+) plasmid (Promega) and transformed into Escherichia coli. Miniprep plasmid DNA was prepared from the transformed bacterium and part of it was digested with EcoRI and YYYCL restriction endonucleases; the clones containing the insertions are arranged sequentially.

The 195-bp DLK cDNA fragment obtained from the emptied PCR was radiolabeled and used to study approximately 1 x 10 ⁶ recombination of Uni-ZAP II (Stratagene, La Jolla, CA), oligo (c1T) -primary cDNA an adult mouse brain library (Holzman, et al., Mol Cell Biol, 1990, 10, 5830-5838). The filters were hybridized to a buffer consisting of 50% formamide, 5 x SSC, 3 x Denhardt solution, 0.25% SDS, 1 mg / ml polyadenyl acid and 200 mg / ml salmon DNA at 42 ° C. The filters were washed once at room temperature in 2 x SSC, 0.2% SDS and twice for 30 min at 65 ° C. Twenty-five identical clones are identified, 10 clones are purified to homogeneity, in vivo cut down according to the manufacturer's protocol and restriction map. The two longest columns (3401 and 3397 bp, respectively, differing only in their 5 'end) are arranged sequentially along the strands along the entire length.

The full-length Noil Xhol fragment of DLK cDNA (3401 bp) was subcloned into a cytomegalovirus carrier based on the eukaryotic expression carrier pcDNA3 (Invitrogen, San Diego, CA) (designated pcDNA3-DLK construction). Subsequently, a curved construction (pFLAG-DLK) of the NHi-Met FLAG epitope (DYKDDDDK) (Sequence ID: 13) is made. PCR is used to amplify cDNA fragments encoding the 5 'Hmd \\\ site, DLK Kozak sequenced sequence, including stimulation of the ATG, FLAG epitope, and DLK cDNA open reading frame sequence extending from nucleotide 88 to internal £ coRI site at nucleotide 758 . (HPLC purified synthesized oligonucleotides used in equimolar amounts: .) Gel purified HindSl and EcoRI-degraded amplified fragments are subcloned into HindSl-EcoRA, 3. and to prepare the pcDNA3-DLK plasmid. The construction follows both the threads to ensure Taq polymerase fidelity and the maintenance of the reading frame.

Example 20: pcDNA3-MLKl

The 5 'portion of MLK1 is obtained from the EST database (access # AA 160611). This clone is a mix between MLKI and another cDNA with unknown identity. It contains an unpublished 5 'sequence of MLK1 together with part of a previously published kinase region of MLK1 (Dorow, et al., Eur. J. Biochem., 1993, 213, 701-710). The MLKI cDNA sequence of the EST clone is as follows: GAATTCGGCA

CGAGAGGACT CGCAGGTGTC CGGCGACGAG GGCTGGTGGA CCGGGCAGCT GAACCAGCGG GTGGGCATCT TCCCCAGCAA CTACGTGACC CCGCGCAGCG CCTTCTCCAG CCGCTGCCAG CCCGGCGGCG AGGACCCCAG TTGCTACCCG CCCATTCAGT TGTTAGAAAT TGATTTTGCG GAGCTCACCT TGGAAGAGAT TATTGGCATC GGGGGCTTTG GGAAGGTCTA TCGTGCTTTC TGGATAGGGG ATGAGGTTGC TGTGAAAGCA GCTCGCCACG ACCCTGATGA GGACATCAGC CAGACCATAG AGAATGTTCG CCAAGAGGCC AAGCTCTTCG CCATGCTGAA GCACCCCAAC ATCATTGCCC TAAGAGGGGT ATGTCTGAAG GAGCCCAACC

TCTGCTTGGT TAGAGTGTTA GTGAATTGGG TACATGATGA TAAGTCCAGC NSAREDSQVS RCQPGGEDPS RAFWIGDEVA HPNIIALRGV

CATGGAGTTT

TCTGGGAAAA CTGTGCAGAT GGCAATTGTT AAC (Sequ. Id.

GDEGWWTGQL CYPPIQLLEI VKAARHDPDE CLKEPNLCLV

GCTCGTGGAG

GGATTCCCCC TGCCAGAGGG CCCATCATCC No: 16). this is

NQRVGIFPSN DFAELTLEEI DISQTIENVR MEFARGGPLN

GACCTTTGAA AGACATCCTG ATGAACTACT ACCGCGACCT broadcasts to:

YVTPRSAFSS IGIGGFGKVY QEAKLFAMLK

RVLSGKRIPP

DILVNWAVQIARGMNYLHDE AIVPIIHRDL KSSN (Sequence ID: 17).

The 3 'portion of MLKI is cloned initially by PCR degradation as previously published (Dorow, et al., Eur. J. Biochem., 1993, 213, 701-710). The cloning protocol I of the 3 ′ portion of MLK1 is, as described above, for MLK2 with the following exceptions. Of the four clones obtained by re-examining the 1.2 kb clone library, three of the four clones represented MLK1. None of the clones fully include the kinase region derived from PCR.

Phages from 1 ml aliquots of amplified libraries (normal human epithelial colon and human T84 cell carcinoma cell line cDNA in 1 Uni-ZAPXR (Stratagene, cat # 937204) were dissolved by suspension in 20 ml water and rapid freezing. of 5 ml of the lysed phage was used as a PCR matrix in two reactions for each library The primers representing the vectors were taken from the nucleotide sequences bordering the cloning sites In the case of T84, the colon cell line library used alternate T3 primers and T7 (Promega) In each reaction The primer is one of the 3-5 'strands of the MLK1 gene, approximately 100bp from the 5' end of the known sequence.The second primer is one of the two vector primers PCR reactions contain IX PCR buffer, 2.5 mM magnesium chloride, 1 U Tag polymerase (all from Bresatec), 0.2 mM dNTP and 0.4 mM each primer in a total of 50 mL The reaction conditions are 60s at

95 ° C, 90s at 52 ° C, 90s at 72 ° C for 30 cycles with 15 min duration in the final cycle. PCR products were cloned and sequenced as described above. The longest branch of library research and

The PCR fragment, which includes an additional MLK1 sequence, was bound together to generate 1.08 kb MLK1 cDNA in pUC 18.

The MLK1 clone from the EST database was obtained in pBluescript media (Stratagene). The MLK1 cDNA from the column library was ligated into the EST clone by digesting the former with Klenow-blunted EcoR1, then excised with Aflll. This isolated fragment was cloned into MLK1 cDNA from EST data cut with Xhol, blunted with Klenow, and cut off with Aflll. This new construct is then excised from pBluescript by degradation with Notl and Apal and bound in pcDNA3-EE also excised with Notl and Apal. All cloning compounds were sequentially checked.

Example 21; E coli expression of GST-MLK3 _kl) pGEXKG-MLK3 (KD) was transformed into E. coli strain BL21 by electroporation. Bacteria containing the plasmid were inoculated into 15 liter Applikon fermenter in 10 liter of the following rich medium: 1.95 g / L K2HPO4, 0.9 g / L KH2PO4, 0.1 g / L ampicillin, 0.3 g / L, © ( NH _{4) 2} SO ₄ , 0.92 g / L MgSO ₄ 7H ₂ 0, 42.7 mg / L Citrate, 21.8 mg / L FeSO ₄

7H ₂ O, 0.5 mL of Pichia micrometals (Higgins, et al., Methods Molecular Biology, 1998, 103, 149-177), 20 g / L of caramino acids, 40 g / L of glycerol, 25.5 mg / L of CaCl ₂ . Bacteria were grown overnight at 800 rpm / 68% dissolved oxygen / 30 ° C until the culture reached OD ₆ oo = 4.4. The production of recombinant protein was induced by the addition of 1 mM isopropyl-β-O-thiogalactoside, with continued fermentation at 25 ° C for up to 6 hours. The bacteria were then recovered by centrifugation and the cell mass was frozen at 20 ° C until purification.

Example 22: Purification of bacterial GST-MLK3kd

Partially purified GST-MLK3 kv was prepared as 100 gm of bacterial cell mass was sonicated in 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol (DTT), pH 7.5 (buffer A). The solution was made 1% with Triton X-100, then stirred on ice for 1 hour. The supernatant, after centrifugation for 45 minutes at 20,000 x g, was mixed for 1 hour on ice with 10 mL of Glutathione Sepharose 4B resin (Pharmacia) at equilibrium in buffer A. The precipitated resin was washed twice with 12.5 x volume buffer A was then washed with 20 mL 100 mM Tris-HCl, 150 mM NaCl, 5 mM DTT (buffer B) containing 20 mM glutathione, pH 7.5. Protein was dialyzed overnight in buffer B and stored in aliquots at -80 ° C.

EXAMPLE 23: FLAG-MLK3 &apos; Viral Expression

GST-MLK3kd

Recombinant rod viruses expressing FLAGMLK3 and GST-MLK3rd expressively were obtained from their respective specific carriers, pFB-FLAG-MLK3 and pFB-GST-MLK3kd, using the VASTO-BAC system (Life Technologies) according to the instructions. Sf21 cell suspension cultures (Vaughn, et al, In Vitro, 1977, 13, 213-217) were grown at 27 ° C / 120 rpm in Grace medium added (Hink, Nature, 1970,226, 466-467) c 10% heat-inactivated fetal bovine serum (FBS). To obtain recombinant FLAG-MLK3, Sf21 cells at a density of 1.5 x 10 ⁶ cells / mL added Grace medium containing 5% FBS were infected with multiplicity of infection (MOI) at 3.1 and harvested at 39 h post-infection. To obtain recombinant GST-MLK3rd, Sf21 cells at a density of 1.5 x 10 ⁶ cells / mL added Grace medium containing 5% FBS were infected with an MOI of 2 and harvested at 41 h after infection. In both cases, the precipitated cells were resuspended in buffer consisting of 10 mM HEPES, 50 mM NaCI, 0.5 mM Pefabloc SC, 5 μΜ pepstatin, 10 μµ / mL aprotinin, 10 pg / mL levpeptin, pH 7.4. The supernatant, after centrifugation for one hour at 147,000 xg, was adjusted to pH 7.4 with a 3 M Tg base and then stored at -70 ° C before purification.

Example 24: Purification of rod-virus GST-MLK3 _K n

Partially purified rod-virus GST-MLK3kd is prepared by chromatography affinity for glutathione. For 10 mL cell extract (26.6 mg total protein), 1 mL of glutathione Sepharose 4B resin (Phatmacia) was equilibrated in 10 mM HEPES, 150 mM NaCl, pH 7.4 (buffer C) was added and the eu protein was allowed to bind for 45 min at 4 ° C. The resin was then washed in a column with 30 column volumes of buffer C, then washed with 5 column volumes of buffer C containing 20 mM glutathione. The recovered final product was dialyzed overnight in buffer C and stored in aliquots at -70 ° C.

Example 25: Purification of rod virus FLAG-MLK3

Partially purified rod-virus FLAG-MLK3 was prepared by antibody-conjugated chromatography. A 15 mL protein from the extract (19.5 mg total protein) with an additional 0.1 M NaCl was bound to a 0.25 mL column of M2 monoclonal FLAG protein antibody bound to agarose resin (Sigma) by reloading (three times total). The resin was equilibrated with 5 column volume washing with 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS), 3 column volume washing with 0.1M glycine, pH 3.5, followed by another 5 column volume washing with TBS. before chromatography. The recombinant protein was initially washed with 5 column volumes of 0.2 mM FLAG protein (N-AspTyr-Lys-Asp-Asp-Asp-Asp-Lys-C) (Sequence ID: 18) in TBS. Store the protein in aliquots at -80 ° C before testing.

Example 26: Dominant negative mutant: Dominant negative mutant of the MLK family blocks death in differentiated PC12 cells after removal of nerve growth factor

The PC-12 cell line derived from rat pheochromocytoma has been widely used as a nerve cell model to investigate the molecular moments leading to neuronal death (for reference, see Troy, et al., Adv. Neurology, 1997, 103- 111). Nerve growth factor (NGF) induces differentiation of PC-12 cells into a sympathetic neuronal phenotype (Greene, Cell Biol., 1978, 78, 747-755). NGF differentiated PC-12 cells depend on NGF for survival and undergo morphologically described apoptotic death upon removal of NGF from the culture medium. A cellular system was developed to determine the effect of members of a mixed kinase family on PC-12 cell death after NGF removal. PC-12 cells underwent transfection with cDNA coding for a dominant negative (DN) mutant of MLK-3 using a Pfx system on a fatty medium as recommended by the manufacturer (Invitrogen, Carlsbad, CA). A permanent transfectant pool expressing DN-MLK-3 was selected using G418 sulfate (Mediatech Inc., Herndon, VA). Approximately 30% of the cells in these funds express DN MLK3 as determined by immunohistochemistry. Cells permanently expressing the mutant kinase were plated on 96-well plates coated with tissue culture polyornitine / laminin (10 µg / ml each in phosphate buffer serum) at a density of 2 x 10 ⁴ cells / well and treated with 100 ng /. ml of NGF for 7 days. The medium containing NGF was removed, the cell monoplast was washed with phosphate buffer serum and the medium containing neutralizing NGF antibody (cat. # N6655; Sigma, St. Louis, MO) at a final dilution of 1: 1000 was removed for 1-5 days . Cell viability was quantified by cell viability assay using the conversion of the tetrazolium salt, MTS, to a colored formazan, which was taken into account when absorbed at 570 nm on CytoFluor 2350 (Millipore, Bedford, MA), as recommended by the manufacturer (Promega, Madison, WI).

Permanent funds expressing DN-MLK-3 were partially rescued from cell death caused by NGF removal (Figure 10).

Example 27: Assay for Enzyme Activity of Recombinant MLK Protein

To show that the MLK protein expressed either in rod or bacterial expression system is enzymatically active, several test specimens may be used. The MLK protein may be a full-length or kinase region expressed either in rod-like virus or in a bacterial expression system. The assay may be based on an antibody such as enzyme-linked immunosorbent assay (ELISA), consistent resolution fluorescence (TRF), or fluorescence polarization (FP). The antibody may be monoclonal or polyclonal with reactivity to a phosphoserine, a phosphotreonine or a phosphospecific substrate. Alternatively, a non-antibody based method may be used, such as gel based radioactive analysis (see Figure 11), trischloroacetic acid (TCA) multiscreen analysis (Figure 13), scintillation expression (SPA) , evaporation column method or phosphocellulose filter test sample (Figure 13). The assay may be for the purpose of monitoring the direct phosphorylation of a substrate or a coupled assay system using downstream kinase kinases in the signaling pathway. The substrate may be a specific substrate such as SEK-1 or a relatively non-specific substrate such as myelin basic protein (MBP).

Example 28: Kinase Assays:

(1) Gel-based radioactive kinase analysis

The kinase activity of MLK-3 is assayed by monitoring the incorporation of P from [γ-P] -ATP into the MLK substrate (eg, lifeless kinase SEK-1; myelin basic protein). A 50-μ1 assay mixture contains Buffer A (20 mM MOPS, pH 7.2, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol), 15 mM MgCl ₂ , 100 μΜ ATP, 10 pCi [γ- ³² Ρ] -ΑΤΡ and 0.1 gg lifeless kinase SEK1 substrate (Stressgen, Inc; Glutathione B-transferase-BEC-1 (GSTSEK-1) bound was released from glutathione-agarose seeds with 10 µM glutathione, pH 8.0) or 25 pg MBP (Sigma Chemical Co.). The reaction is initiated by the addition of an MLK protein (kinase region or preparation containing both full-length and kinase region) or control protein. The mixture was incubated for 30 min at 30 ° C. At the end of the reaction, a 2x reducing assay buffer was added. The mixture was refluxed for 5 min, loaded on either a 12% SDS-PAGE gel (using MBP as substrate) or 8% gel (SEK-1 as substrate). After electrophoresis, the gel was dried. Quantification of phosphate incorporation into the substrate, SEK-1, was performed on a Molecular Dynamics Phosphorimager (Sunny Phosphorus Molecular Dynamics Apparatus) (Sunnyvale, CA). The results of the experiments show the enzymatic activity of rod-viral expressing MLK-3 (FLAG-labeled full-length or GST-labeled kinase region) using lifeless kinase GST-SEK-1 or MBP as a substrate are shown in Figures 11A and 11B.

(2) Western Blot Analysis

The kinase activity of rod-viral expressing MLK-3 was verified by immunoassay staining. The 20-μ1 mixture analyzed contains Buffer A, 15 mM MgCl ₂ , 100 μΜ ATP and 0.1 μg lifeless kinase SEK-1 substrate. The reaction was allowed to proceed for 30 min at 30 ° C, then quenched sharply with 10 μΐ 4 times with reducing sample buffer. The proteins were separated on an 8% Trisglycine gel and electrophoretically transferred to an Immobilon PVDF membrane. The membrane was incubated with phosphospecific SEK-1 (Thr223) antibody (New Engln Biolabs, Inc.), followed by horseradish peroxidase-labeled goat anti-rabbit IgG (Bio-Rad). Determining of

immunoreactive bands were performed by enhanced chemoluminescence (Amersham). Phosphorylation of GST-SEK-1 lifeless kinase by the FLAG-MLK-3 protein (rod-like virus containing both full-length and kinase regions) is shown in Figure 12.

(3) Multiple analysis of trichloroacetic acid (TCA) precipitation

The kinase activity of the bacterially expressed GST-MLK-3 kinase region was evaluated using Millipore multiple trichloroacetate (TCA) assay in a plate, as described by Pitt, et al., J. Biomol. Screening, 1996, 1, 47-51). Analyzes were performed in 96-well, multiple-pore solid Millipore plates. Each 50-μ1 mixture analyzed contains 20 mM Hepes, pH 7.4, 20 mM MgCI ₂ , 20 mM MpCl ₂ , 2 mM DTT, 0.1 mM Na ₃ VO ₄ , 1 pCi [γ-Ρ ³² ] ATP and 30 pg MBP substrate . The reaction was started by the addition of MLK protein and allowed to proceed for 15 min at 37 ° C. The reaction was stopped with 25 µl of 50% TCA. The plates were allowed to equilibrate for 30 min at 4 ° C, then washed with ice-cold 25% TCA. A scintillation cocktail was added to the plates and radioactivity was determined using a Wallac MicroBeta 1450 PLUS scintillation counter. The protein dose response against the formation of ³² P-labeled MBP is shown in Figure 13.

(4) Phosphocellulose filter assay

The kinase assay was performed in a 50-p1 reaction mixture containing 20 mM Hepes, pH 7.4, 20 mM MgCl ₂ , 20 mM MpCl ₂ , 2 mM

DTT, 0.1 mM Na ₃ VO4, 1 pCi [γ-Ρ ³² ] ATP and 30 pg MBP. The reaction was started by the addition of MLK protein and allowed to proceed for 15 min at 37 ° C. The reaction was stopped with 75 μ 75 of 75 mM phosphoric acid. An aliquot of the cooled solution was loaded directly onto the phosphocellulose membrane (Pierce). In addition, a 96-well phosphocellulose multiscreen plate (Millipore) may be used. The membranes were washed with 75 µM H ₃ PO ₄ . The bound ³² P-labeled phosphorylated MBP was washed in collector tubes by the addition of 1 M sodium hydroxide. Radioactivity was determined by counting Cerenkov in a Beckman scintillation counter (Somerset, NJ). The formation of phosphorylated MBP with increasing concentration of bacterially expressing GSTMLK-3 kinase region is shown in Figure 13.

Example 29: Assay to determine the binding of compounds to a mixed MLK family

K-252a (Compound III-3; see Table 4), an indolocarbazole metabolite of the Nocardia species, binds to various serine / threonine and tyrosine kinases (Angeles, et al., Anal. Biochem, 1996, 236, 49-55; Knight, et al., Anal. Biochem., 1997, 247, 376-381). Tritium containing K-252a ligand was used to analyze binding to human full-length mixed MLK-3 from crude preparation of rod-viral infected cells. [ ³ H] K-252a was specifically labeled with tritium at the 3 and 9 positions by contact with NEN Research products (Billerica, MA) and had a specific activity of 40 Ci / mmol. Binding reactions were performed in 1 ml in a 96-well plate. The reaction mixture contains 50 mM MOPS buffer, pH 7, 150 mM NaCl, 5 mM MPSCl ₂ , 1 mg / ml BSA, 1% ο

DMSO and 0.25 nM [H] K252a. Samples were performed in triplets at a concentration of 5 µg / ml crude rod viral derivative MLK-3.

Non-specific binding is defined as binding in the presence of unlabeled 1.2 µM K252a and deduced from the general binding to obtain the specific binding. At this dilution, 12-15% of the total number were not specifically bound to protein and 75-85% were specifically bound to MLK-3 (Figure 14). All experiments were performed for 2 hours at 4 ° C. [ ³ H] K252a / MLK-3 complexes were collected on GF / C Whatman filters using Brandel mice, washed with cold MOPS / NaCI buffer and counted on a Wallac Micro Beta counter. A saturation coupling experiment was performed to obtain Ka for K252a. An example of the results of one of these experiments is shown (Figure 14). Ka 0.89 nM (Confidence Range: 0.2 to 1.5 nM) was obtained.

Example 30: Intact Cell Assays (A) Cos 7 Overexpression System

Materials

K-252a and derivatives of this compound are provided by KyowaHakko Kogyo Co. Ltd. (Tokyo, Japan) (Kaneko et al., 1997). The compounds were dissolved in a cell culture fraction of dimethyl sulfoxide (DMSO) and stored in the dark at 4 ° C. All dilutions of the compounds were made in Dulbecco modified Eagle medium (DMEM) containing 1% bovine serum albumin. Hemagglutinating antibody (HA) is supplied by BAbCC (Richmond, CA). The AP-1 (with jun) substrate was supplied by Promega (Madison, WI). [γ- ³² Ρ] ΑΤΡ (6000 Ci / mmol) is supplied from Amersham (Arlington Heights, IL).

Cos7 cell culture

Green monkey kidney Cos7 cells were obtained from ATCC, Rockville Maryland (CRL 1651) and kept in DMEM containing 10% bovine serum, 2 mM glutamine, 1 mM pyruvate, 50 U / ml penicillin / streptomycin at 37 ° C in 10% CO ₂ , 90% atmospheric air. Cos7 cells were separated for passage by the addition of Q.25% trypsin.

(1) Overexpression of MLK family members and JNK1 in Cos7 cells

Cos7 cells were plated at 80% confluence and transfection with 2 µg of each cDNA construct using lipofectamine as recommended by the supplier (Gibco BRL, Gaithersburg, MD). Full-length human MLK-3, MLK-2, or mouse DLK or partially human MLK-1 cDNA, as described above, and full-length human JNK1 A-terminus hemagglutinin kindly supplied by J. Silvio Gutkind (NIH , Bethesda, MD) were subcloned into a pcDNA3 carrier (Invitrogen, San Diego, CA). After 48 hours of transfection, cells were treated with 0.025% DMSO or 500 nM of the indicated compounds for 2 hours, followed by lysis in 0.4 ml of Triton buffer (1% Triton X-100, 50 mM sodium chloride, 10 mM Tre (pH 7.6), 0.1% bovine serum albumin, 30 µM sodium pyrophosphate, 50 mM sodium fluoride, 20 µg / ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate). JNK activity was evaluated by lysis by immunoprecipitation / kinase assay as described below.

(2) Whole cell immunoprecipitation and kinase assay

Cos 7 cell lysate was measured for protein concentration using a Micro BCA kit from Pierce (Rockford, IL), and equal amounts of protein were immunoprecipitated with HA antibody for 1 hour at 4 ° C. The immunoprecipitates were precipitated by centrifugation in a microfuge centrifuge for 20 s, resuspended in Triton buffer, washed 3 more times, followed by final washing in kinase buffer (20 mM Hepes pH 7.4, 20 mM MgCl ₂ , 2 mM dithio , 0.1 mM sodium vanadate). The immunoprecipitate was resuspended in kinase buffer containing 1 μΜ ATP and 5 pCi [γ-P] ATP and substrate (1 pg / sample of AP-1) and incubated for 15 min at 30 ° C. The kinase reaction is stopped by the addition of the reducing sample buffer (Laemmli, Nature 1970: 227; 680-685). Samples were heated to 80 ° C for 5 min and loaded onto 10% SDS polyacrylamide gels. Proteins are separated by electrophoresis. The body was dried and quantified for radioactivity in the AP-1 substrate on a Molecular Dynamics Phosphorimager (Sunnyvale, Ca.). The results of experiments where MLK-3, MLK-2 and DLK are co-expressed with HA-JNK1 and incubated in the absence or presence of K-252a are shown in Figures 15A and 15B. In contrast, a derivative of starting compound K-252a, called Compound III-3 (see Table 4), which is a more selective kinase inhibitor, does not interact with the JNK pathway activated by another MAPKICK, a higher branch of JNK, MEKKI (Figure 15C).

(B) Analysis of whole cell reporting for JNK-activated MLK

Attempts to produce clones that are systemically expressive of the MLK family are unsuccessful, suggesting that MLKs overexpression may affect cell survival (Bergeron et al., Biochem. Biophys.Res.Commun., 1991,231, 153- 155; Nagata, et at. EMBO J. 1998, 17, 149-158). Therefore, a cell line containing a genetically engineered kinase expression system of interest may be needed to develop an entire cell assay to track MLK-induced biochemical events. For example, a PC-12 cell line infected with a tetracycline controlled transactivator. When cells are further infected with a gene of interest derived from a tetO-inducing promoter, the expression of this gene is tightly controlled by tetracycline in the medium (Shocket, et al. Proc. Natl. AcadSci. USA, 1995, 92, 6522 ).

In order to quantify MLK activation, the phosphorylation of substrates in the lower clone, such as MEK4, JNK, or c-jun in complex test forms as described above, can be measured. Another approach to quantify MLK activation in whole cells is to use enzyme activity reporting, such as a c-jun luciferase messaging system, which can be commercially available through the PathDetect ™ system (Stratagene, LaJolla, CA). In this system, tetracycline-inducing cell line is infected with two plasmids. One plasmid constructively expresses fusion of the eJun NH ₂ terminal transactivation region with the GAL4 DNA binding region (cJun-DBD fusion protein). The other plasmid carries the firefly luciferase coding sequences extracted by five double iterations of the GAL4 binding side. Upon activation of MLK, the bottom branch of the JNK substrate, the cJun-DBD fusion protein, is phosphorylated, binds to the GAL4 binding sites, and induces transcription of the luciferase gene. Luciferase is readily assayed in cell lysates by the addition of a substrate (Promega, Madison, WI) and chemoluminescence measurement.

Example 31: Relationship between MLK Family Inhibition by Motoneuron and Cortical Survival

Survival of spinal cord cultures of rats enriched with motoneurons

Spinal cord dissection of embryonic embryos of Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) at embryonic age (E) 14.5-15. Cells only from the ventral part of the spinal cord are dissociated and further enriched for motoneurons by centrifugation at a metrizamide gradient of 6.5% step as described above (Henderson al., 1993) and analyzed for purity by low staining affinity neurotropic receptor antibody (IgG-192, Boehringer-Mannheim) (data not shown). The cells were seeded onto 96-well plates pre-coated with poly-1-ornithine and laminin (5 µg / ml each) at a density of 6x10 ⁴ cells / cm ² in chemically defined serum medium without N2 (Bottenstein, et al., 1979 , supra). In order to separate the effects of accession from those of survival, compounds were added to the cultures after an initial period of 1-3 hours of accession. Neuronal survival was assessed after 4 days using calcein AM (Molecular Probes, Eugene, OR) in fluorometric viability analysis (Boryczko-Coyne, et al., 1993, supra). Microscopic counting of neurons correlates directly with relative fluorescence values. Briefly, the culture medium was serially diluted in DPBS (Dulbeccos phosphate buffer serum) and then a final concentration of 6 [mu] M calcein AM was added to each 96-well. The plates were incubated for 30 min at 37 ° C, followed by serial dilution of washes in DPBS. The fluorescence signal was read using a Millipore plate reader (Cytofluor 2350) with excitation = 485 nm and emission = 538 nm. For each tile, the mean background obtained from the calcein AM cells but not containing cells was extracted from all values. The linearity of the fluorescence signal is determined precisely by the concentration and incubation time for the variation in cell density in these experiments. An example of percent survival over control of motoneurons in the presence of test compounds at 250 nM is shown in Table 3.

Survival of cortical neurons

The cerebral cortex is dissected from rat embryos at 18 embryonic days and is enzymatically digested to form a single cell suspension. Cells were seeded at a density of 1.56 x 105 / cm on poly-ornithine / laminin coated 96 well tissue culture plates in serum free Neural Basal medium containing B27 additives. The plates were coated with a solution of poly-ornithine / laminin (8 µg / ml each) made in PBS for at least 2 hours at 37 ° C. At 5-7 days in vitro, cortical neurons were exposed to AB25-35 (20 µM) either in the presence or in the absence of test compounds. AB25-35 (Sigma, St. Louis, MO) stock solutions (1mM) were prepared in deionized-distilled sterile H ₂ O. Relative neural survival was determined 48 hours after protein attachment using lactate dehydrogenase (LDH) separation, such as Plasma membrane integrity / cell viability indicator. LDH was measured using the Cytotoxicity Detection Kit (Boehringer-Mannheim, Indianapolis, IN) in accordance with the manufacturer's instructions. Data are shown as percent inhibition of LDH release on cultures treated with AB25-35 alone.

Table 3

Cortical neurons Motoneurons Cos-7 Cells Cos-7 Cells Cos-7 Cells Cos-7 Cells formula Survival% Control at 250 nM Survival% Control at 250 nM % JNK 500 nM DLK% JNK Inhib. @ 500 nM MLK3% JNK Inhib. @ 500 nM MLK2% JNK Inhib. @ 500 nM MLK1% JNK Inhib. @ 500 nM III ¹ 46, 56 300% 65% 63, 73 99, 98 89, 67 97.96 III ² 47.80 315% 88% 36,22, 42 94.94 69, 44 92.64 I ³ 22,54 177 88% 20,25 94.93 0 79.29 t 29.39 165% 97% 58.13, 52,8 84.92, 90 0 63.38

The compound has formula III, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ ; and R is OH.

The compound has the formula III, wherein Ζι and Ζ ₂ are Η; X is CO ₂ CH ₃ ; R 1 and R ₂ are CH ₂ SCH ₂ CH ₃ ; and R is OH.

The compound has the formula I wherein A _b A ₂ , R _b R ₃ , R ₅ and R ₆ are H; B] and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R 4 is CH ₂ CH ₂ (2-Pyridyl); and X is CH _2.

^The compound has the formula I, wherein A _b L ₂ , R 1, R ₃ , R 5 and R 5 are H; IN! and B ₂ together represent O; R ₂ is H; R 4 is CH ₂ CH ₂ (2-Pyrimidinyl); and X is

CH ₂ .

Example 32: Immunoprecipitation of endogenous JNK activity from motoneuron cultures in the absence or in the presence of indolocarbazoles or condensed pyrrolocarbazoles

Purified motoneurons were placed in a 6 x 10 ⁴ cells / cm ² plate in 16 mm diameter wells. The cells were allowed to fix for 2 hours before treatment. Cells were treated with either 0.0125% DMSO or 500 nM compound for 2 hours in N2-specific medium. The cells were then washed with ice-cold phosphate-buffered serum, followed by lysis in 0.4 ml of Triton buffer as described above in Example 30. The lysate from the motoneuron culture was normalized to cell number and immunoprecipitated with JNK1 antibody (cat. # Sc -474) supplied by Santa Cruz Biotechnology (Santa Cruz, CA). The JNK activity of the immunoprecipitates was assayed in the presence of P-ATP and c-jun substrate as described above. The inhibitory activity profile of the 4 test compounds was compared in motoneurons and in Cos7 cells, overexpressing either DLK, MLK-1, MLK-2, or MLK-3 (Table 3).

Example 33: Correlation between MLK3 Inhibition of JNK-Induced Activity in Cos7 Cells and Cholinacetyltransferase Activity in Primary Embryonic Cultures

To determine whether inhibition of the JNK pathway regulated by these kinases correlates with neurotropic compounds, we evaluate the effect of the compound on JNK activity in Cos7 cells overexpressing HA-JNK and MLK3. After a 48 hour transfection period, cells were incubated with the compounds at 500 nM for 2 hours, followed by cell lysis. The lysate was immunoprecipitated and kinase activity was measured as described above. The results are reported as percent inhibition of the control sample, where control is JNK activity in the presence of DMSO. As can be seen in Table 4, most compounds that are active in spinal cord and / or basal forebrain ChAT activity are protein inhibitors of MLK-3 activation of JNK.

Table 4

Effect of indolo- and indeno-carbazoles on JNK activity in Cos7 cells overexpressing MLK3

Choline acetyltransferase activity % Inhibition of JNK Activity (Medium) Compound The spinal cord Basal forebrain MLK3 in Cos7 cells PM ¹ + + 84 ΪΪΕ? + + 96 PEZ ³ + + 94 E1 ⁴ + + 93 E2 ⁵ + + 85 EU ³ + + 93.5 E4 ⁷ - + 95 E5 ⁸ - + 97 E6 ⁹ - + 58 E7 ¹⁰ - + 85.5 W-4 ¹¹ - + 66 IIE5 ¹² - + 96 IIE7 ¹³ - + 54 E8 * + - 89

W-8 ¹⁵ + - 94 W-9 ¹⁶ + + 98.5 III-10 ¹⁷ + - 78 I-9 ¹⁸ + - 88 I-10 ¹⁹ + -. 94 III-II ²⁰ - - 92.5 1-11 ²¹ - + 33 I-12 ²² - - 11 1-13 ²ⁱ - - 1

'A compound of formula III, wherein Ζι and Z ₂ are Η; X is CO ₂ CH ₃ ; R 1 is NHCONHC ₂ H ₅ ; R ₂ is CH ₂ CH ₂ (2-Pyridyl); and R is OH.

^{2 A} compound of formula III, wherein Ζι and Z ₂ are Η; X is CO ₂ CH _3; R 1 and R ₂ are CH ₂ OCH ₂ OCH ₂ CH ₃ ; and R is OH.

A compound of formula III wherein Z ₁ and Z ₂ are Η; X is CO ₂ CH ₃ ; R 1 and R ₂ are CH ₂ SCH ₂ CH ₃ ; and R is OH.

^{4 A} compound of formula I wherein A _b A ₂ , R _b R ₃ and R 4 are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; R 5 and R ₆ are OCH ₃ ; and X is CH ₂ .

^A compound of formula I wherein A _b A ₂ , R _b R ₃ , R ₅ and R ₅ are H; IN! and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R 4 is Br and X is CH ₂ .

^{6 A} compound of formula I wherein A _b A _2; R _b R ₃ , R ₅ and R ₆ are H; B] and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R 4 is CH ₂ CH ₂ (2-Pyridyl); and X is CH _2.

^A compound of formula I wherein A _b A ₂ R _b R ₃ , R 4, R ₅ and R 6 are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; and X is CH ₂ .

Q

A compound of formula I wherein A _b A ₂ , R _b R ₃ , R 4, R 5 and R 4 are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ CH ₂ OH; and X is CH ₂ .

^A compound of formula I wherein A _b A ₂ R _b R ₂ , R ₃ , R 4, R ₅ and R 4 are H; IN; and

B ₂ together represent O; and X is S.

^A compound of formula I wherein A _b A ₂ , R _b R ₃ , R 4, R 5 and Re are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ CH ₂ NHCO (4- (OH) Ph); and X is CH ₂ .

^{11 A} compound of formula III wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ (CH ₂ ) ₄ CH ₃ ; and R is OH.

A compound of formula III, wherein Z _b Z ₂ and R 1 are H; R ₂ is CH ₂ OH; X is CO ₂ CH ₃ ; and R is OH.

A compound of formula III wherein 7L 1 and Z ₂ together form = O; R 1 and R ₂ are Br; X is CO ₂ CH ₃ ; and R is OH.

^A compound of formula I wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and R 6 are H; B _t and B ₂ together represent O; R ₂ is H; R 4 is CH ₂ CH ₂ (2-Pyrimidinyl); and X is CH ₂ .

^A compound of formula III wherein Z 1 and Z ₂ are H; R1 is Br; R ₂ is I; X is CO ₂ CH ₃ ; and R is OH.

^A compound of formula III wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ ; and R is OH.

A compound of formula III wherein Z 1 and Z ₂ are H; R 1 and R ₂ are CH ₂ CH ₂ SCH ₃ ; X is CO ₂ CH ₃ ; and R is OH.

A compound of formula I wherein A _b A ₂ , R _b R ₂ , R ₃ , R ₅ and R ₆ are H; B _t and B ₂ together represent O; R4 is CH ₂ CH ₂ (2-Pyridazinyl); and X is CH ₂ .

^A compound of formula I wherein A _b A ₂ , R _b R ₃ , R ₅ and R ₆ are H; B ₁ and B ₂ together represent O; R ₂ is H; R 4 is CH ₂ CH ₂ (2-Pyridyl); and X is CH ₂ .

Compound of Formula III, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ ; and R is OCH ₃ .

A compound of formula I wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and R ₆ are Η; Bi and B ₂ together represent O; R ₂ is (CH ₂ ) ₃ -> 1 -C (= O) -3,5-dihydroxyphenyl; and X is CH ₂ .

A compound of formula I wherein A _b A ₂ , R _b R ₃ , R. _b R ₅ and R ₆ are Η; Βι and B ₂ together represent O; R ₂ is benzoyl; and X is CH ₂ .

A compound of formula I wherein A _b A ₂ , R _b R ₂ , R ₃ , R ₅ and R _f are Η; Βι and B ₂ together represent O; R4 is CH = CH-C ^ N; and X is CH ₂ .

Example 34: Gel Shift Analysis for MLK Activation:

Activation of MLKs may result in the induction of transcription of c-jun, leading to an increase in c-Jun protein. The increased amount of c-Jun protein can be measured by standard analysis, defined as gel displacement analysis. Gamer, et al., Nucleic Acids Res., 1981, 9, 3047-3060, which is incorporated herein by reference in its entirety. Radiolabeled double DNA oligomers encoding the c-Jun DNA binding side were incubated with nuclear cell extract followed by acrylamide gel electrophoresis and quantification of radiolabeled DNA shifted to slow motility. This represents that portion of DNA that is a bound c-Jun protein and is directly proportional to the amount of c-Jun protein in the extract.

Activation of MLKs may also induce phosphorylation of c-Jun. This can be determined using antibodies that specifically recognize the phosphorylated form of the protein in determining systems, such as, for example, Western blots or ELISA.

Example 35: Survival of chicken embryonic neurons

Materials

The medium of Leibovitz L15, glucose, sodium bicarbonate, trypsin and antibiotics are from Gibco. A muscle extract is prepared as described (Henderson, et al., Nature, 1983, 302, 609-611, which is incorporated herein by reference in its entirety). All other reagents are from Sigma unless otherwise indicated.

Cell culture

Motoneurons (embryonic day 5.5) were isolated by immunological method according to the procedure described in Bloch-Gallego, et al., Development, 1991, 111, 221-232, which is incorporated herein by reference in its entirety, with modifications as described in Weng, et al.,

NeuroReport, 1996, 7, 1077-1081, which is incorporated herein by reference in its entirety. The purified motoneurons were seeded on 35 mm tissue culture plates (Nunc) pre-coated with poly-DLornitin and laminin (1 gg / ml, Upstate Biotech). The culture medium was 15 L with sodium bicarbonate (22.5 mM), glucose (20 mM), progesterone (2x10 'M), sodium selenite (3x10'M), conalbumin (0.1 mg / ml), insulin (5 pg / ml). , penicillin-streptomycin and 10% heat-inactivated horse serum. The muscle extract was supplemented to 30 gg / ml. Compound III-3 was prepared as a 4mM stock solution in DMSO and stored in a light-protected place at 4 ° C. The final concentration of DMSO in the treated and control culture was 0.125%.

The paravertebral sympathetic ganglion (SG; embryonic age 12 days (E 12)), dorsal root ganglion (DRG; E9) and ciliary ganglion (CG; E8) are dissected from chicken embryos of the indicated embryonic age, as described in Lindsay, et al., Dev. Biol., 1985, 112, 319-328, which is incorporated herein by reference in its entirety. After trypsinization and dissociation, the suspensions of nerve cells were plated on polyornithine-laminin coated plates in Ham F14 culture medium supplemented with 10% horse serum. Survival factors were added immediately after placement of the plates at the following concentrations: Nerve Growth Factor (NGF), 20 ng / ml; ciliary neurotropic factor (CNTF), 10 ng / ml. The cultures were maintained at 37 ° and 5% CO ₂ in a humidified environment.

Cell counting

Neurons were placed in tiles in 35 mm Nune culture. Selected areas from each plate containing together about 10% of the test surface for the presence of the bright cell phase immediately after insertion and again after 48 hours to estimate survival rates. Cell survival was confirmed by vivid trypan blue staining (not shown).

Intact DRG

The ganglia were placed in 96 well plates pre-coated with poly-1-ornithine and laminin (5 µg each / ml phosphate buffer serum) in N2-free serum (Bottenstein, et al., Proc. Natl. Acad. Sci. USA, 1979, 76, 514-517, which is incorporated herein by reference in its entirety) containing 0.05% bovine serum albumin (BSA) and maintained for 48 hours at 37 ° and 5% CO ₂ in humidified environment. The treated ganglia received either 250 nM Compound III-3 or 20 ng / ml NGF 2 hours after plating.

Compound III 3 promotes the survival of chicken embryonic peripheral neurons in a concentration-dependent model

Retrieval of NGF from dissociated cultures of E9 sensory neurons from dorsal root ganglia (DRG) and E12 neurons of sympathetic ganglia (SG) subjected them to PCD for 48 hours. This is prevented by adding Compound III-3 to the culture medium during NGF withdrawal. AT 1 μΜ, Compound III-3 kept 94% of SG neurons and 890% of DRG neurons alive after 48 hours (NGF-treated: SG 65%, DRG 66%). Similarly, Compound III-3 promotes the survival of 76% of CNTF-dependent ciliary ganglion (CG) neurons after 24 hours (67% treated with CNTF control). In the presence of 10% serum, the survival effects of Compound III-3 are concentration-dependent, with a plateau reaching about 1 μΜ for all three neuronal populations (Fig. 16A-C). Surviving neurons show greater axon growth with thicker and more arched axons compared to control cultures. (Fig. 17 EH). After four days, the stimulating activity of survival was still intact: DRG: Compound III-3 52% treated with growth factor control 41%; SG: Compound III-83 83%, NGF-treated 55%; CO: Compound III-58 (CNTF-treated 50% (50%). At

optimum conditions, cultures can be maintained with Compound

W-3 for one week or more (not shown).

Compound III 3 promotes the survival of chicken embryonic motoneurons in a concentration-dependent model

Cultured chicken motoneurons can survive and expand processes in the presence of a muscle extract while they die quickly in the absence of it. In our experiments, after 48 hours, 65% of the motoneurons survive in the presence of muscle extract, as opposed to 14% of the untreated controls. In serum-free conditions, the survival effect of Compound III-3 was maximal at 300 nM and slightly higher (79%) than that induced by muscle extract. In this system, the dependence of the survival effect of Compound III-3 on concentration is different from that of peripheral neurons, because concentrations of Compound III-3 above 300 nM show a progressively diminished effect (Fig. 16A). This may indicate a particular sensitivity of the motoneurons to a particular side of Compound III-3 activity. Morphologically, the motoneurons rescued with Compound III-3 show cell bodies in a clear phase and are able to produce long axons that appear slightly thicker than those induced by muscle extract (Fig. 17). After four days in culture, 56% of the motoneurons were alive with Compound III-3, compared with 42% with muscle extract. At 300 nM, Compound III treated neurons survive in vitro for at least one week (not shown).

Compound III 3 promotes the growth of axons from the intact ganglia of the dorsal root

The results of the above experiments indicate that Compound III-3 not only promotes the survival of embryonic neurons of the peripheral and central nervous systems, but also leads to strong axonal growth. Many of these growths turn out to be thicker than those extracted in the presence of growth factors (compare Fig. 17 A-D with Fig. 17 E-H).

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This effect was also observed in axonal growth obtained from intact cultures of embryonic dorsal root ganglia in the presence of 250 nM Compound III-3 (Fig. 18C). The axons grow both in response to NGF (Fig. 18B) and to compound III-3; these extracted through NGF are much finer and branched than those grown in the presence of Compound III-3, which appear to be thick and can be bundled.

Example 36: In vivo treatment

Evolutionarily regulated motor neuron death in a chicken embryo

This example is described in detail in Glicksman, et al., J. Neurobiol., 1998, 35, 361-370, which is incorporated herein by reference in its entirety. On E6, a window is made into the chicken egg shell (Spafas, Preston, CT) and a filler (5% Solutol ™ HS 15, polyethylene glycol 660 Hydroxyestellate; BAS) is added directly to the vascularized chorioallantoid membrane once a day from E6 to E9. , Ludwigshafen, Germany (in phosphate-buffered serum, pH 7.2)) or the specific dose of Compound III-3 in vehicle, as described in Oppenheim, et al., Science, 1991, 251, 1616-1617, which is incorporated herein by reference in its entirety). The embryos were killed on E10 and their spinal cords were separated, fixed in a solution of Satou (10% glacial acetic acid, 60% absolute ethanol, 30% chloroform), treated for serial paraffin sections and stained with thionine. Each 20th slice of lumbar segments 1 through 8 was counted according to previously described criteria (Clarke, et al., Methods in Cell Biology: Cell Death, 1995, Schwart & Osborne, Eds., Academic Press, New York, pp. 277-321, which is incorporated herein by reference in its entirety).

Evolutionally regulated motor neuron death in neonatal rats

Sprague-Dawley pregnant rats of indefinite duration were obtained from Harlan Laboratories (Indianapolis, IN). Female small rats were injected subcutaneously (SC) daily over the target perineal muscle, with Compound III-3 in 5% Solution ™ HS 15 or with filler, starting on the first day of birth (P1) and continuing for 5 days (P5 ). At P10 or P60, the small ones are decapitated, blood is collected in heparinized capillary tubes and the spinal cord region containing the sexually dimorphic spinal nucleus of the bulbous cavernous (SNB) and the perineal area containing the bulbous cavernus eleusor (LA). ) are dissected after serum / formalin perfusion of the animals. The spinal cord region containing the SNB is then fixed, flooded with Paraplast, cut into ΙΟμπι and stained with Cresylecht violet (Nordeen, et al., Science, 1985, 229, 671-673, which is incorporated herein by reference in its entirety). Motor neurons were counted at X500 in consecutive sections from 5 lumbar to 1 sacral spinal cord region, as described above (Nordeen, et al., Supra). Microscopic numbering is done on coded sections by an observer who is "blind" to the treated groups. The number of motor neurons is adjusted according to cell size and slice thickness (Konisgsmark, Contemporary Research Methods in Neuroanatomy, Nauta & Ebbesson, Eds., 1970, SpringerVerlag, New York, pp.315-340, incorporated herein by citation in its entirety) and statistical analysis is a one-way analysis of variation (ANOVA). The perineal muscle was then fixed, decalcified, flooded with Paraplast, sectioned at 10 pm and stained with Milligan's Trichrome. Using bright microscopy (X250), BC and LA muscles in normal females and females treated with Compound III-3 (405 animals / group) were positively identified in both their localization and the presence of layered fibers. Muscle tissue outline was monitored by modified sections using a projection microscope (62.5) and the cross section was measured using a digitized pad and a computer morphometric system (Sigmascan, Jandel Scientific). The muscle volume was calculated by treating the entire cross-sectional area and by the thickness of the slice and adjusted for the percentage of the structural sample.

The collected blood was centrifuged for 5 min at room temperature; the plasma was then removed and frozen at -20 ° C. Serum testosterone levels (6-7 animals / group) are measured by radioimmunoassay following the procedures outlined in Wingfield, et al., Steroids, 1975, 26, 311-327, which is incorporated herein by reference in its entirety.

Motor neural dedifferentiation in adult rats induced by axonotomy

The left sublingual nerve was excised in the neck of an adult female Sprague-Dawley rat (120-180 g), anesthetized with Neumbutol and 50 μΐ of Compound III-3 or its filler (5% Solutol ™ HS 15) applied to a piece Gelfoam ™ (AJ Buck, Owings Mills, MD) then wrapped around the proximal end of the severed nerve. After 7 days, the animals were intoxicated and perfused with 4% paraformaldehyde in Sorenson buffer, 0.07 M phosphate, pH 7.2. The brain stem is removed and layer slices 40 cm thick are made on a cryostat (Chiu, et al., NeuroReport, 1994, 5, 693-696, which is incorporated herein by reference in its entirety). Every fifth slice was continued for ChAT immunohistochemistry as described above (Chiu, et al., J. Comp. Neurol., 1993, 328, 351-363, which is incorporated herein by reference in its entirety) using 1: 350 dilution of an anti-ChAT monoclonal antibody obtained from Chemicon. Cells that stain clearly above the background are counted in stained sections; the number of numbered cells is expressed as the ratio of the number of ChAT-immunoreactive cells on the axonotomized side of the nucleus of the sublingual nerve to the number of immunoreactive cells on the control (non-injured) side.

Compound III-3 rescues rat embryonic motor neurons from cell death by apoptosis in vitro and inhibits the signaling mechanism leading to JNK1 activation in these cells (Maroney, et al., J. Neurosci., 1998, 18, 104-111, which is incorporated herein by reference in its entirety). To determine possible in vivo activity, Compound III-3 is evaluated in two models of evolutionarily regulated programmed motor neuron death and a model of axonotomy-induced dedifferentiation in age-related motor neurons. In chickens, approximately 50% of spinal motor neurons undergo PCD during E5-10 (Hamburger, et al., J. Neurosci., 1982, I, 38-55; Purves, et al., Body u Brain: A Trophic The Theory of Neural Connections, 1988, Harvard University Press, Cambridge, MA are both incorporated herein by reference in their entirety). Administration of Compound III-3 to the chorioallantoid membrane during this period prevents dose-dependent death of the motor neuron (Fig. 19). Forty percent of motor neurons that would normally die are removed at the two highest test doses (2.3 and 7 pg / zmeBHo), while 25% of motor neurons are delivered at lower doses (1.2 and 1.8 pg / mieBHo). (Fig. 19).

During early perinatal life in female rats (late embryonic stage up to 4 postnatal days (PN), more than 50% of motor neurons in the SNB are eliminated by PCD (Breedlove, J. Neurobiol., 1986, 17, 157-176. which is incorporated herein by reference in its entirety.) In males, motor neurons in these nuclei innervate the inferior musculature of the penis, including the sexual reflexes. Sun and LA muscles innervated The administration of testosterone to female small leads to a full number of muscle SNB motor neurons (Nordeen, et al., supra) and prevents BC and LA muscle atrophy (Waiman, et al., Endocrinology, 1941, 29, 955-978, incorporated herein by reference in its entirety.) Daily subcutaneous administration of Compound III-3 (PN 1-5) in female rats significantly attenuates the death of motor neurons (Fig. 20A). -H occurs at two doses (0.5 and 1 mg / kg per day). At maximum effective doses of 0.5 and 1 mg / kg per day, administration of Compound III-3 resulted in a 70% increase in the survival of motor neurons, equivalent to the effect of testosterone (Fig. 20A). Compound III-3 did not alter the plasma levels of testosterone in the treated female. Radioimmunoassay of plasma testosterone levels in the 1 mg / kg / day group resulted in a slight difference when compared with the vehicle control group (0.016 ± 0.008 ng / mL and 0.029 ± 0.015 ng / ML, respectively, of the mean ( SEM)).

To determine whether treatment with Compound III-3 was effective in maintaining the survival of motor neurons for a long time, females were treated with Compound III-3 (0.5 and 1 mg / kg / day) for the same time period PN (1-5) (from the first to the fifth fasting day). Half of the animals in the filler groups and both treatment groups were sacrificed on the tenth postnatal day (PN10). The remaining animals were then maintained without further treatment with Compound III-3 until sacrificed at 60 postnatal day (PN6O). As observed previously (Fig. 20A), treatment with Compound III-3 resulted in a 70% increase in the survival of motor neurons (Fig. 20B). In addition, 100% of these rescued motor neurons can be identified morphologically 55 days after the last treatment with Compound III-3 (Fig. 20B). Delay with Compound

W-3 of the death of motor neurons during the neonatal period allows the survival of motor neurons during the age period.

Despite the clear demonstration and devastating effects of motor neuron loss in 25 adult humans, diseases such as lateral amyotrophic sclerosis, motor neurons in adults in most motor nerve trauma models are life-sustaining. However, axonal trauma indeed leads to morphological (Oppenheim, et al., Supra) as well as biochemical changes (Oppenheim, et al., Supra; Rende, et al., J. Comp. Neurol., 1992, 319, 285298, which is incorporated herein by reference in its entirety; Chiu, et al.,

J. Comp. Neurol., 1993, 328, 351-363, which is incorporated herein by reference in its entirety) in adult motor neurons, which may resemble a degenerative change preceding death in disease in degenerative motor neurons. One example of this kind of change comes from the axonomy of the sublingual nerve that innervates the tongue. Unilateral interruption of this nerve in the adult rat results in the loss of 95% of ChAT-immunoreactive sublingual motor neurons in the ipsilateral nuclei after 7 days (Chiu, er al., NeuroReport, 1994, 5, 693-696, which is incorporated herein by reference in its entirety). The loss of ChAT immunoreactivity is not permanent. Four weeks after the axonotomy, 100% of motor neurons restore control levels of ChAT immunoreactivity (Borke, et al., J. Neurocytol., 1993, 22, 141-153, which is incorporated herein by reference in its entirety). ChAT immunoreactivity in contralateral sublingual motor neurons was unaffected (Chiu, et al., Supra) (Fig. 21 and Table 5).

When administered in Gelfoam ™ to the proximal end of the sublingual nerve, Compound III-3 attenuates in a dose-dependent manner the decrease in ChAT immunoreactivity in ipsilateral sublingual motor neurons, evaluated 7 days after axonotomy.

The maximum effective dose (50 μg) resulted in 40% more ChATimmunoreactive motoneurons compared to axonotomized untreated controls (Fig. 21B and Table 5). There is a disproportionate dose dependency with both lower and higher doses leading to greater survival than in the untreated control group, but less than that obtained at 50 pg. As with the SNB model, there was no associated weight loss, mortality, or destruction of large tissue in these animals at any of the doses tested.

In three separate in vivo models of motor neuron degeneration, Compound III-3 showed neuroprotective activity: evolutionarily regulated PCD of lumbar motor neuron division in embryos (Fig. 19), androgen-sensitive death of postnatal SNB motor neurons (Fig. 20). ) and axonotomy-induced loss of a functional marker, ChAT, in sublingual motor neurons of an adult (Fig. 21 and Table 5). Compound III-3 is effective when administered peripherally by subcutaneous injection, applied topically to the severed end of the nerve or directly above the chicken embryonic chorioallantoid membrane. Contrary to the parent molecule K-252a, Compound III-3 is approximately five times more potent in mediating the survival of cultures enriched with motor neurons (data not shown) and does not show inhibitory activity against trkA tyrosine kinase and several serine threonine kinases (Maroney et al., Supra, Kaneko, et al., J. Med Chem., 1997, 40, 1863-1869, which is incorporated herein by reference in its entirety).

Table 5

Effect of compound III-3 on cholinacetyltransferase immunoreactivity in axonotomized hypoglossal motor neurons

ChAT-positive motor neurons Treatment η Experimental / Control % Medium / Group filler 2 20/544 3.68 4.01 19/437 4.35 3.6 μg W-C 2 55/420 13.10 12.84 * 72/572 12.59 25 pg W-H 2 95/597 15.91 19.01 * 142/642 22.12 50 pg W-H 2 188/484 38.84 41.34 * 278/637 43.85 100 pg W-H 4 465/920 50.54 32.61 * 235/784 29.98 178/770 23.12 182/679 26.80 200 pg W-C 2 99/461 21.48 24.96 * 159/559 28.44 Simulated impact 2 350/335 104.48 101.24 292/298 98.00

To the proximal end of the sublingual nerve, Compound III-3 or a filler is added to the foam gel immediately after its cutting. Animals were sacrificed after 7 days and sequential sections were made through the sublingual nuclei, and every fifth slice was stained with immune antiChAT antibodies. List the ChAT-positive neurons in

100 ipsilateral (experimental) and contralateral (control) sides of the nuclei * p <0.05, statistically significant compared with the control group of the vehicle treated animals.

The MLK-3 pathway inhibitor shows efficacy in vivo and blocks phosphorylation in the lower MLK-3 in the MPTP model

MPTP is administered at a dose (40 mg / kg) that results in loss of striatal dopaminergic endings and cell bodies in the nigra substance. Tyrosine hydroxylase is used as a marker of dopaminergic nerve endings in the nigra substance. Systemic administration of Compound III-3 reduced the loss of tyrosine hydroxylase immunoreactive neurons of a nigra substance after a MPTP lesion (Fig. 22a; Saporito et al., 1999). Since Compound III-3 is a known MLK3 inhibitor, activation of a lower MLK3 substrate is measured in an MPTP-treated mouse. The levels of phosphorylated MCC4 were measured using a phospho-MCC4 specific antibody (New England Biolabs, Beverly, MA) that recognizes the monophosphorylated form of MCC4 either by immune staining (Fig. 22b) or by ELISA (Fig. 22c). Administration of MPTP increased the levels of phosphorylated ICP4 in the nigra substance up to 5-fold above control levels (Fig. 22b). Peak increases occur 4 hours after MPP administration and coincide with peak CNS levels of MPP. The MPTP-mediated phosphorylation of MKK4 is attenuated by pretreatment with 1-deprenyl, indicating that these phosphorylation phenomena are affected by MPP ⁺ (Fig. 22c). Moreover, the phosphorylation of MKK4 was partially inhibited by pretreatment with Compound III-3 at a dose (1 mg / kg) which protected against MPTP-induced dopaminergic loss (Fig. 22c). These data indicate that MPTP (MPP ⁺ ) activates MKK4, a lower substrate of MLK3. Moreover, these data show that known

101 inhibitor of MLK3, inhibits the activation of this kinase pathway in vivo.

Example 37: Inflammation

Induction of IL-1 and TNF-α by LPS in THP-1 cells and the effect of indolocarbazoles and pyrrolocarbazoles on their induction

Immune system cells are selected because many kinases are involved in the regulation of numerous immunological functions, eg, induction of cytokine synthesis and induction of the biological response of cytokines. One message (Hambleton, et al., Proc. Natl. Acad Sci. USA, 1996, 93, 2774-2778, which is incorporated herein by reference in its entirety) indicates that the treatment of monocyte cell line derivatives with LPS causes rapid activation of JNK activity. When monocytes come into contact with bacterial endotoxins, such as lipopolysaccharide (LPS), they produce the inflammatory cytokines IL and TNF-α. Inhibition of the production of these two types of cytokines may be a useful treatment for certain inflammatory diseases of the immune system. These cytokines can be readily measured by commercial ELISA kits. We create experiments to determine (1) whether indolo- and fused pyrrolocarbazoles can inhibit the synthesis of IL-1 and TNF-α in our monocytic THP-1 cell line, (2) whether JNK is activated by LPS in THP-1 cells and (3) whether the activation of JNK by LPS can be inhibited by indolo- and fused pyrrolocarbazoles.

Experimental procedures

THP-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. LPS (E.coli serotype 0111.84, TCA extracted) was supplied from Sigma and dissolved in PBS. ELISA kits for IL1 and TNF-α assay are supplied by Boerhinger-Mannheim and assays are performed on THP-1 culture media as specified by the manufacturer. Standard curves according to the guidelines were obtained for each analysis.

102

The experiments were carried out in 12 culture plates with 1 or 2 ml of THP-1 cells at 4 X 10 ³ cells / ml. IL-1 and TNF-α were induced by the addition of LPS to the culture medium and the medium was harvested at different times for cytokine analysis. The cells were removed by centrifugation and the supernatants were frozen at -70 ° C until analysis. To reduce the value of the experiments, they were performed in duplicate cultures and the duplicate supernatants were extracted after centrifugation. Each supernatant extracted is evaluated twice. Stock solutions of indolo- and fused pyrrolocarbazoles in 100% DMSO were diluted to the desired concentrations either in medium containing 10% bovine fetal serum or in medium containing 0.5 mg / ml BSA. Unless explicitly mentioned, the compounds are added to THP-1 cells 1 hour before the addition of LPS.

JNK activity assays are performed after immunoprecipitation of the JNK protein from lysed THP-1 cell extract. The precipitated THP-1 cells were lysed on ice for 15 minutes in 540 μΐ Frac buffer (10 mM Tris-Hcl, pH 7.5, 50 mM NaCI, 30 μΜ sodium pyrophosphate, 1 mg / ml BSA, 1% Triton-X- 100). The extract was centrifuged for 10 minutes at 14K and 5 μΐ JNK antibody (Santa Cruz) was added to the supernatant. The extract was rotated for 60 minutes at 4 ° C, 75 μΐ of the washed A Sepharose protein (20% w / v in Frac) was added and the extract rotated for another 30 min to bind the antibody complex to the A Sepharose protein. The Sepharose protein was washed twice with Frac buffer, once with 20 mM Hepes, pH 7.6, 20 mM MgCl ₂ , 2 mM DTT, then incubated for 15 min at 30 ° C in 30 μΐ kinase buffer (20 mM hepes, 20 mM MgCl ₂ , 2 MM DTT, 1 μβ mixed with jun and 2 μΜ ΑΤΡ-γ- ³² Ρ, 2 μθ The reaction was quenched by the addition of 10 μΐ 4X SDS gel filling buffer, warmed for 3 min at 80 ° C and proteins were analyzed for

103

10% SDS gel. The gel was dried, exposed to a Phosphorimager plate, and radioactive regions analyzed for Phosphorimager.

The results of the initial experiments show that LPS at 2 pg / ml gives the maximum production of IL-1, and this concentration of LPS is used in all subsequent experiments. The minimum time after the addition of LPS for maximum cytokine production is determined by taking aliquots of the assay medium at different times after the addition of LPS. The first experiment showed that both IL-1 and TNF-α attenuated maximal production at least 5 hours after the addition of LPS. Since the earliest collection time in the first experiment was 2.4 hours, the second experiment was performed in the collected media starting 15 min after LPS collection. The results of this experiment, where only TNF-α was analyzed, indicated that it attenuated the maximal yield 3 hours after the addition of LPS. No significant TNF-α was detected in the medium until 90 min after the addition of LPS.

The rapid attainment of maximum yield indicates a very tight regulation of 2 cytokine synthesis - rapid synthesis and rapid re-regulation. Cell cultures were treated for 30 min before the addition of LPS or with Actinomycin D, an RNA synthesis inhibitor or cycloheximide, an inhibitor of protein synthesis. The medium was collected 3 hours after the addition of LPS and TNF-α was analyzed. Both new RNA and new protein synthesis are required for the induction of TNF-α, since TNF-α is not detected in the middle of cells treated with one of the inhibitors. The following experiments are performed to determine whether Compound III-3 would inhibit the induction of IL-I and TNF-α. Compound III-3 inhibited the induction of both IL-1 and TNF-α by IC50 values, respectively, of 267 nM and 139 nM. The results of these experiments were obtained with cells in medium containing 10% bovine embryo serum. Because the analyzes of spinal cord tissue and basal forebrain tissue for

104

the neurotropic activity of the compounds was performed in serum-free medium (500 gg / ml BSA), determination of IC50 values for inhibition of IL1 and serum-free TNF-α was of interest. When THP-1 cells were treated with Compound III-3 in serum-free medium (500 gg / ml BSA), IC50 was reduced 10-fold from 269 nM to 23 nM. Unless otherwise stated, all experiments performed thereafter are performed in serum-free medium. Inhibition of Compound III-3 by the induction of IL-1 and TNF-α in THP-1 cells suggests that Compound III-3 can be used as a therapeutic in the treatment of pathological conditions caused by the production of these cytokines in amounts of larger than normal. Septic shock is such a condition. Septic shock is caused by an increase in gram negative bacteria in the bloodstream, which in turn leads to the release of large amounts of endotoxin, LPS. LPS then initially stimulates monocytes and macrophages to produce large amounts of IL-1 and TNF-α, which then causes massive tissue destruction and, in many cases, death.

Several compounds have been tested for their ability to inhibit TNF-α and compared with their ability to inhibit JNK. The results are shown in Table 6.

Table 6

TNP-1 Cells Overexpression of MLK3 in Cos 7 cells Compound TNF-a IC50 nM JNK% in. 500 nM JNK% in. 500 nM III-1 49.5 93.5 83.8 W-W 29 93 94 1-2 > 5000 78.5 85 1-3 366 80.5 93.7

105

1-4 75.5 79.5 95 1-5 514 89 97.2 1-6 817.5 77.5 57.8 1-7 1009 74 85.5 W-4 462.5 81 66 W-5 4 84.5 96 W-7 590.5 11.5 54 W-8 11.5 51 94 III-10 4298 48 78 1-10 4500 62 94 III-11 686 51 92.5

Effect of Compound III-3 on the induction of IL-2 in Jurkat cells Experiments were performed to determine whether Compound III-3 inhibited the induction of IL-2 in Jurkat cells.

Experimental procedures

Jurkat cells are grown in RPMI 1640 medium supplemented with 10%

embryonic bovine serum. TNF-α is from Promega and anti CD3 and anti CD28 antibodies are from Pharmigen. Jurkat experiments were performed in 200 μΐ in a 96 well plate. IL-2 was measured with an ELISA kit supplied by Boehringer Mannheim. Antibodies to CD3 and CD28 were allowed to bind to the plastic mass of the 96 well plate (18 hours in PBS) before Jurkat cells were added. The cells were treated with compound 1 hour before being added to the antibody coated plate. Antibodies to CD3 and CD28 have been used to activate the T cell receptor and to induce IL-2. IL-2 was released from Jurkat cells between 6 and 24 hours after the start of induction (Fig. 23A). The effect of Compound III-3 (treatment with Compound III-3 1 hour before induction) on the induction of III-2 was then evaluated (Fig. 23B). The concentration of Compound III-500 500 nM inhibits the induction of IL-2 by more than 80%

106 (FIG. 23B). A more extensive dose-response experiment was performed with Compound III-3 and Compound 1-4, which gave IC ₅₀ values for Compound III-139 139M and Compound 1-4 207nM (Fig. 23C).

It is claimed that each patient, application, and printed publication mentioned in this patent are incorporated herein by reference in their entirety.

Those skilled in the art will appreciate that numerous changes and modifications can be made to preferred aspects of the invention without departing from the spirit of the invention. It is claimed that all such variants fall within the scope of the present invention.

Claims (4)

1. A method of identifying a compound that modulates the activity of a complex kinase protein line and promotes cellular survival, characterized in that it comprises the steps of: (a) contacting a cell containing a complex kinase protein line with a compound; b) determining whether a compound reduces the activity of a complex kinase protein line; c) determining whether a compound promotes cellular survival. The method of claim 1, wherein the protein is selected from the group consisting of complex kinase line 1, complex kinase line 2, complex kinase line 3, leucine zigzag kinase, double leucine zigzag kinase and complex kinase line 6 . The method of claim 2, wherein the cell contacts in vitro. The method of claim 2, wherein the cell contacts in vivo. 5. The method of claim 2, wherein the activity of a protein is determined by measuring the activity or the phosphorylation step of a protein substrate. 6. The method of claim 5, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρ38δ, MEK1, MEK2, MKK3, MKK4 (SEK1) , MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEH-3. The method of claim 2, wherein the activity of a protein is determined by measuring the activity of a substrate of a protein, the amount of substrate of a protein, or the mRNA encoding a given substrate of a protein. The method of claim 2, wherein the activity of a protein can be determined by an in vitro kinase assay or binding assay. 9. The method of claim 2, wherein aiding cell survival is determined by using cells at risk of dying and comparing the amount of living cells that have been in contact with a compound with the amount live cells that have not been in contact with a compound. The method of claim 9, wherein the cells are primary embryonic cells of motoneurons. The method of claim 9, wherein said cells are overexpressed with respect to a protein of a complex kinase line. 12. The method of claim 2, wherein aiding cell survival is determined by observing a decrease in apoptosis. 13. The method of claim 2, wherein the cell is a neural cell. The method of claim 2, wherein the cell is involved in a neurodegenerative disease. 15. A method of identifying a compound that modulates the activity of a complex kinase protein line and promotes cellular survival, characterized in that it comprises the steps of: (a) contacting a cell containing a complex kinase protein line with a compound; b) determining whether a compound enhances the activity of a complex kinase protein line; and c) determining whether a compound promotes cell death. The method of claim 15, wherein the protein is selected from the group comprising complex kinase line 1, mm complex kinase line 2, complex kinase line 3, leucine zigzag kinase, double leucine zigzag kinase and complex kinase line 6. The method of claim 16, wherein the cell contacts in vitro. 18. The method of claim 16, wherein the cell contacts in vivo. The method of claim 16, wherein the activity of a protein is determined by measuring the activity of a substrate of a protein. 20. The method of claim 19, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρ38δ, MEK1, MEK2, MKK3, MKK4 (SEK1) , MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEH-3. 21. The method of claim 16, wherein the activity of a protein is determined by measuring the activity of a substrate of a protein, the amount of a protein, or the mRNA encoding a protein. 22. The method of claim 16, wherein the activity of a protein can be determined by an in vitro kinase assay or binding assay. 23. The method of claim 16, wherein aiding cell survival is determined by using cells at risk of dying and comparing the amount of living cells that have been in contact with a compound with the amount of living cells that have not been in contact with a compound. The method of claim 23, wherein said cells are primary embryonic cells of motoneurons. 25. The method of claim 23, wherein said cells are overexpressed with respect to a protein of a complex kinase line. 26. The method of claim 16, wherein the promotion of cellular survival is determined by observing an increase in apoptosis. 27. The method of claim 16, wherein the cell is a neural cell. The method of claim 16, wherein the cell is involved in a neurodegenerative disease. 29, A method of modulating the activity of a complex kinase protein line comprising contacting a protein or cell containing a protein with a compound of the formula: characterized in that: ring B and ring F, independently of each and together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring, where from 1 to 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring, where or 1) one carbon atom is replaced by an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced by sulfur and nitrogen atoms, oxygen and nitrogen atoms or two nitrogen atoms; and 3) three carbon atoms are replaced by three nitrogen atoms; R ¹ is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heteroarylalkyl; u 6) -C (= O) R ⁹ , wherein R ⁹ is selected from the group consisting of alkyl, aryl and heteroaryl; b) -CR ¹⁰ , wherein R ¹⁰ is selected from the group consisting of H and alkyl having from 1 to 4 carbons; r) -C (= O) NH ₂ , -NR ⁿ R ¹² , - (CH 2) ^{p N} R ⁿ R ¹² , - (CH 2) _P OR ¹⁰ , O (CH 2) POR ¹⁰ and -O (CH 2) _p NR ⁿ R ¹² , wherein p is from 1 to 4; and where or 1) R ⁿ and R ¹² are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbons; or 2) R ⁿ and R ¹² together form a linking group of the formula - (CH 2 -X 4 CH 2b 2 -, wherein X ¹ is selected from the group consisting of - O -, - S-and-CH ₂ .; R ² is selected from the group consisting of H, alkyl having from 1 to 4 carbons, -OH, alkoxy having from 1 to 4 carbons, -OC (= O) R ⁹ , OC (= O) NR ⁿ R ¹² , -O (CH 2) PNR ¹¹ R ¹² , -O (CH 2) _p OR ¹⁰ , substituted or unsubstituted arylalkyl having from 6 to 10 carbons and substituted or unsubstituted heteroarylalkyl; R ³ , R ⁴ , R ⁵ and R ⁶ are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF 3, -ΝΟ 2 -, -OH, OR ⁹ , -O (CH 2) _p NR ⁿ R ¹² , -OC (= O) R ⁹ , -OC (= O) NR ² R ⁷ , -OC (= O) NR ⁿ R ¹² , O (CH 2) pOR ¹⁰ , -CH2OR ¹⁰ , -NR ⁿ R ¹² , -NR ¹⁰ S (= O) ₂ R ⁹ , -NR ¹⁰ C (= O) R ⁹ ; 6) -CH ₂ OR ¹⁴ , wherein R ¹⁴ is an amino acid residue after the hydroxyl group of the carboxyl group is removed; b) -NR ¹⁰ C (= O) NR ¹¹ R ¹² , -CO ² R ² , -C (= O) R ² , -C (= O) NR ⁿ R ¹² , CH = NOR ² , -CH = NR ⁹ , - (CH 2) _p NR ¹¹ R ¹² , - (CH ₂ ) _P NHR ¹⁴ or -CH = NNR ² R ^2A , 2 a 2 where R is the same as R; r) -S (O) _Y R ² - (CH 2) PS (O) _Y R ⁹ , -CH 2 S (O) ₇ R ¹⁴ , where y is 0, 1 or 2; e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons and alkynyl having from 2 to 8 carbons, where 1) each alkyd, alkenyl or alkynyl group is unsubstituted; or 2) each alkyd, alkenyl or alkynyl group is substituted by from 1 to 3 groups selected from the group consisting of aryl having from 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy, hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy, alkoxy, alkoxy, alkoxy, alkoxy Cl, Br, I, -CN, -NO ₂ , -OH, -OR ⁹ , -X ² (CH 2) _p ^N R ⁿ R ¹² , X ² (CH 2) _p C (= O) NR ¹¹ R ¹² , -X ² (CH2) pOC (= O) NR ⁿ R ¹² , -X ² (CH 2) _P CO ₂ R ⁹ , X ² CH ² S (O) YR ⁹ , -X ² (CH 2) _p NR ¹⁰ C (= O) NR ¹¹ R ¹² , -OC (= O) R ⁹ , -OCONHR ² , -O-tegrahydropyranyl, -NR ⁿ R ¹² , -NR ¹⁰ C (= O) R ⁹ , NR ¹⁰ CO2R ⁹ , -NR ¹⁰ C (= O) NR ⁿ R ¹² , -NHC (= NH) NH 2, NR ¹⁰ S (O) 2R ⁹ , -S (O) YR ⁹ , -CO ₂ R ⁹ , -C (= O) NR ⁿ R ¹² , -C (= O) R ² , -CH ² OR ¹⁰ , -CH = NNR ² R ^2A , -CH = NOR ² , -CH = NR ⁹ , -CH = NNHCH (N == NH) NH 2, -S (= O) ₂ NR ² R ^2A , P (= O) (OR ¹⁰ ) ₂ , -OR ¹⁴ and a monosaccharide having from 5 to 7 carbons, where each the hydroxyl group of the monosaccharide is independently or unsubstituted or 3 substituted by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons; X ² is O, S or NR ¹⁰ ; R and R are each independently selected from the group consisting of H, alkyl having from 1 to 4 carbons, alkoxy having from 1 to 4 carbons substituted MNHM1 an or unsubstituted arylalkyl having from 6 to 10 carbons substituted or unsubstituted heteroarylalkyl, - (CH ₂ ) _P OR ^{1 ()} , - (CH 2) POC (= O) NR ¹¹ R ¹² and (CH 2) _p NR ^h R ¹² , or R ⁷ and R ⁸ together form a linking group of formula CH 2 -X ³ -CH 2 -, wherein X ³ is X ² or a bond; m and η are independently 0, 1 or 2; Избран is selected from the group consisting of -O-, -S-, -N (R ¹⁰ ) -, -N ⁺ (O ') (R ¹⁰ ) -, N (OR ¹⁰ ) -and-CH ₂ -; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, C (= O) -, -CH (OR ¹⁰ ), -N (R ¹⁰ ) -, -N (OR ¹⁰ ) -, -CH (NR ⁿ R ¹² ) -, -C (= O) N (R ¹⁷ ) -, N (R ¹⁷ ) C (= O) -, -N (S (O) y R ⁹ ) -, -N (S (O) yNR ¹ R ¹² ) -, -N (C (= O) R ¹⁷ ) -, -C (R ¹⁵ R ¹⁶ ) -, -N ⁺ (O ') (R ¹⁰ ) - , -CH (OH) -CH (OH) -, and CH (O (C = O) R ⁹ ) CH (OC (= O) R ^9A ) - ₅ where R ^9a is the same as R ⁹ ; R ¹⁵ and R ¹⁶ are independently selected from the group consisting of H, -OH, C (= O) R ¹⁰ , -O (C = O) R ⁹ , hydroxyalkyl and -CO ₂ R ¹⁰ ; R ¹⁷ is selected from the group consisting of H, alkyl, aryl and heteroaryl; A ¹ and A ² are independently selected from the group consisting of Η, Η; H, OR ² ; H, -SR ² ; H, N (R ² ) ₂ ; and a group wherein A ¹ and A ² taken together form a unit selected from the group containing = O, = S and = NR; B ¹ and B ² are selected from the group consisting of Η, Η; H, -OR ² ; H, SR ² , H, N (R ² ) ₂ ; and a group wherein B ¹ and B ² together form a unit selected from the group containing = O, = S and = NR; Provided that at least one of the pairs A and A, or B and B, form = 0. 30.Methods for modulating the activity of a complex kinase protein line comprising contacting a protein or cell containing a protein with a compound of the formula: inHnwac characterized in that: Z 1 is H and Z ₂ is H or Z 1 and Z ₂ together form = O; R 1 is selected from the group consisting of H, Cl, CH ₂ SO ₂ C ₂ H ₅ , Br, CH ₂ S (CH ₂ ) ₂ NH ₂ , CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH ₂ , P-C 4 H 9, nhconhc ₆ h ₅ , nhconhc ₂ h ₅ , CH ₂ SC ₂ H ₅ , CH ₂ SC ₆ H ₅ , N (CH ₃ ) ₂ , ch ₃ , CH ₂ OCONHC ₂ H ₅ , NHCO ₂ CH ₃ , CH ₂ OC ₂ H ₅ , CH ₂ N (CH ₃ ) ₂ , OH, O-n-propyl CH = NNH-C (= NH) NH ₂ , CH = NN (CH ₃ ) ₂ , CH ₂ S (CH 2) 2 NH-nC ₄ H 9, CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH ₃ ; CH = N— / \ 4CH3 R ₂ is selected from the group consisting of H, Br, Cl, I, CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH ₃ and CH ₂ OH; X is selected from the group consisting of H, CH ₂ OH, CH ₂ 1CH-SerineH, CO ₂ CH ₃ , CONHC ₆ H ₅ , ch ₂ nhco ₂ c ₆ h ₅ , CH ₂ NHCO ₂ CH ₃ , ch ₂ n ₃ , CONHC ₂ H ₅ , CH ₂ HCG-Glycine, CON (CH ₃ ) ₂ , -CH ₂ NHCO ₂ -, CONH ₂ , CONHC ₃ H ₇ , CH ₂ NH-CepnH, CH ₂ SOCH ₃ , CH = NOH. CH ₂ XH-G1rolin, CH ₂ CH ₂ (2-Pyridyl), CH = NNHC (= NH) NH ₂ , CONH (CH ₂ ) ₂ OH, CH = NNHCONH ₂ , CH ₂ OCOCH ₃ , -CH ₂ OC (CH ₃ ) ₂ O-, ch ₂ sc ₆ h ₅ , CH ₂ SOC ₆ H ₅ , CO ₂ n-hexyl, CONHCH ₃ and CO ₂ (CH ₂ ) ₄ CH ₃ ; and R is selected from the group consisting of OH and OCH ₃ . The method of claim 30, wherein Z 1 and Z ₂ are Η, X is CO ₂ CH ₃ , R 1 is NHCONHC ₂ H ₅ , R ₂ is CH ₂ CH ₂ (2-Pyridyl) and R eon. 32. The method of claim 30, wherein Ζι and Ζ ₂ are Η, X is CO ₂ CH ₃ , R 1 and R ₂ are CH ₂ OCH ₂ OCH ₂ CH ₃ and R is OH. 33. The method of claim 30, wherein Z, and Z ₂ are Η, X is CO ₂ CH ₃ , R 1 and R ₂ are CH ₂ SCH ₂ CH ₃ and R is OH. 34. The method of claim 30, wherein Z _b Z ₂ , R 1 and R ₂ are Η, X is CO ₂ CH ₃ ; and R is OH. 35. The method of claim 30, wherein Z _b Z ₂ , R 1 and R ₂ are X, X is CO ₂ (CH ₂ ) ₄ CH ₃ and R is OH. 36. The method of claim 30, wherein Ζι, Ζ ₂ and R 1 are H, R ₂ is CH ₂ OH, X is CO ₂ CH ₃ and R is OH. 37. The method of claim 30, wherein Ζι and Z ₂ are H, R 1 and R ₂ are H ₂ S (3- (1,2,4-triazine)), X is CO ₂ CH ₃ and R is OH. The method of claim 30, wherein Z 1 and Z ₂ are H, R 1 is Br, R ₂ is I, X is CO ₂ CH ₃ ; and R is OH. 39. The method of claim 30, wherein Z ₂ and Z ₂ are H, R 1 and R ₂ are CH ₂ CH ₂ SCH ₃ , X is CO ₂ CH ₃ and R is OH. 40. The method of claim 30, wherein Z _b Z ₂ , R 1 and R ₂ are Η, X is CO ₂ CH ₃ and R is OCH ₃ . The method of claim 30, wherein Ζι and Z ₂ together form = O, R 1 and R ₂ are Br, X is CO ₂ CH ₃ and R is OH. 42. Methods for modulating the activity of a complex kinase protein line comprising contacting a protein or cell containing a protein with a compound of the formula: A »A * characterized in that: Zi is H and Z ₂ is H or Ζι and Ζ ₂ together form = O; R1 is H or Br; R ₂ is H; R ₃ is H, CH ₂ CH = CH ₂ , CH ₂ CH ₂ CH ₂ OH or CH ₂ CH ₂ CH ₂ - __O R ₄ is H, CH ₂ CH = CH ₂ or CH ₂ CH ₂ CH ₂ OH. The method of claim 42, wherein R 1, R ₂ , R 4, Ζι and Z ₂ are H and R ₃ is CH ₂ CH = CH ₂ . The method of claim 42, wherein R 1 is Br and R ₂ , R ₃ R 4, 4ι and Ζ ₂ are Η. The method of claim 42, wherein R 1, R ₂ , Ζι and Z ₂ are H and R ₃ and R 4 are CH ₂ CH = CH ₂ . The method of claim 42, wherein R 1, R ₂ , R ₃ , Ζι and Z ₂ are H and R 4 is CH ₂ CH = CH ₂ . 47. The method of claim 42, wherein R 1, R ₂ , Ζι and Z ₂ are H and R ₃ and R are CH ₂ CH ₂ CH ₂ OH; or R _b R ₂ , R4, Ζι and Ζ ₂ are / - \ H and R ₃ is CH ₂ CH ₂ CH ₂ --__ O 48. A method of identifying a compound that can be used in the treatment of a neurodegenerative disorder comprising contacting a cell or cell extract containing a complex kinase protein line with the compound and determining whether the compound reduces the activity of a complex kinase protein line. The method of claim 48, wherein the protein is selected from the group consisting of complex kinase line 1, complex kinase line 2, complex kinase line 3, leucine zigzag kinase, double leucine zigzag kinase and complex kinase line 6 . 50. The method of claim 49, wherein the cell contacts in vitro. 51. The method of claim 49, wherein the cell contacts in vivo. 52. The method of claim 49, wherein the activity of a protein is determined by measuring the activity or the phosphorylation step of a protein substrate. 53. The method of claim 52, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρ38δ, MEK1, MEK2, MKK3, MKK4 (SEK1) , MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEH-3. 54. The method of claim 49, wherein the activity of a protein is determined by measuring the activity of a substrate of a protein, the amount of substrate of a protein, or the mRNA encoding a substrate of a protein. 55. The method of claim 49, wherein the activity of a protein can be determined by an in vitro kinase assay or binding assay. 56. The method of claim 49, wherein said cells are primary embryonic cells of motoneurons. 57. The method of claim 49, wherein the cells are overexpressed with respect to a protein of a complex kinase line. 58. The method of claim 49, wherein the cell is a neural cell. 59. The method of claim 49, wherein the cell is involved in a neurodegenerative disease. 60. A method of identifying a compound that can be used in the treatment of inflammation, comprising contacting a cell or cell extract containing a complex kinase protein line with the compound and determining whether the compound reduces the activity of a complex kinase protein line. 61. The method of claim 60, wherein the protein is selected from the group comprising complex kinase line 1, complex kinase line 2, complex kinase line 3, leucine zigzag kinase, double leucine zigzag kinase, and complex kinase line 6 . 62. The method of claim 61, wherein the cell contacts in vitro. 63. The method of claim 61, wherein the cell contacts in vivo. 64. The method of claim 61, wherein the activity of a protein is determined by measuring the activity or stage of phosphorylation of a substrate of a protein. 65. The method of claim 64, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, ρ38α, ρ38β, ρ38γ, ρ38δ, MEK1, MEK2, MKK3, MKK4 (SEK1) , MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEH-3. 66. The method of claim 61, wherein the activity of a protein is determined by measuring the activity of a substrate of a protein, the amount of substrate of a protein, or the mRNA encoding a substrate of a protein. 67. The method of claim 61, wherein the activity of a protein can be determined by an in vitro kinase assay or binding assay. The method of claim 61, wherein the cells are primary embryonic cells of motoneurons. 69. The method of claim 61, wherein the cells are overexpressed with respect to a protein of a complex kinase line. 70. The method of claim 61, wherein the cell is a neural cell. 71. The method of claim 61, wherein the cell is involved in inflammation. 72. A method for treating mammals having a neurodegenerative disorder comprising administering to the mammal a compound that inhibits a complex kinase protein line in a pharmaceutically acceptable salt or diluent. 73. The method of claim 72, wherein the compound has the formula wherein E ¹ and E ² , independently and each together with the carbon atoms to which they are attached, form either an unsaturated 6-membered carbocyclic aromatic ring in which one to three carbon atoms may be replaced by a nitrogen atom (s) ); or an unsaturated 5-membered carbocyclic aromatic ring; wherein either one carbon atom is replaced by an oxygen, nitrogen or sulfur atom; or two carbon atoms are replaced by a sulfur and nitrogen atom, or an oxygen and nitrogen atom; A ¹ and A ² together represent O and B ¹ and B ² together represent O; R ¹ is H, alkyl having from 1 to 4 carbon atoms (including), aryl, arylalkyl, heteroaryl and heteroarylalkyl; COR ⁹ , wherein R ⁹ is alkyl having from 1 to 4 carbon atoms (including) or aryl, preferably phenyl or naphthyl; -OR ¹⁰ , wherein R ¹⁰ is alkyl having from 1 to 4 carbon atoms; -CONH2, -NR ⁷ R ⁸ , - (CH 2) n NR ⁷ R ⁸ , where η is an integer from 1 to 4 (inclusive); or -O (CH 2) _n NR R and or R and R are independently H or alkyl having from 1 to 4 carbon atoms (including), or R ⁷ and R ⁸ are joined together to form a linking group of the general formula - (CH ₂ ) ₂ -X ¹ - (CH 2) 2-, wherein X ¹ is O, S or CH 2; R ² is H, -SO ² R ⁹ ; -CO ² R ⁹ , -COR ⁹ , alkyl having from 1 to 8 carbon atoms (including), preferably alkyl having from 1 to 4 carbon atoms (including), alkenyl having from 1 to 8 carbon atoms (including) , preferably alkenyl having from 1 to 4 carbon atoms (including), or alkynyl having from 1 to 8 carbon atoms (including), preferably alkynyl having from 1 to 4 carbon atoms (including), or a monosaccharide having from 5 to 7 carbon atoms (inclusive), wherein each hydroxyl group of the monosaccharide is independently or unsubstituted or replaced by H, alkyl having from 1 to 4 carbon atoms (including), alkylcarbonyloxy having from 2 to 5 carbon atoms (including) or an alkoxy group having from 1 to 4 carbon atoms (including), and or any alkyl having from 1 to 8 carbon atoms ( alkenyl having from 1 to 8 carbon atoms (including), or alkynyl having from 1 to 8 carbon atoms (including) is unsubstituted; or each alkyl having from 1 to 8 carbon atoms (including), alkenyl having from 1 to 8 carbon atoms (including), or alkynyl having from 1 to 8 carbon atoms (including) is independently substituted by 1-3 aryl having from 6 to 10 carbon atoms (including), preferably phenyl or naphthyl, heteroaryl, F, Cl, Br, I, -CN, -NO ₂ , OH, -OR ⁹ , -O (CH 2) n NR ⁷ R ⁸ , -OCOR ⁹ , -OCONHR ⁹ , O-tetrahydropyranyl, NH 2, -NR ⁷ R ⁸ , -NR ¹⁰ COR ⁹ ; -NR ¹⁰ CO2R ⁹ , -NR ¹⁰ CONR ⁷ R ⁸ , -NHC (= NH) NH 2, -NR ¹⁰ SO ² R ⁹ , -S (O) y R, where R ¹¹ is H or alkyl having from 1 to 4 carbon atoms , aryl having from 6 to 10 carbon atoms, preferably phenyl or naphthyl or heteroaryl and y is 1 or 2; -SR ¹¹ , CO2R ⁹ , -CONR ⁷ R ⁸ , -CHO, COR ⁹ , -CH2OR ⁷ , -CH = NNR ⁿ R ¹² , -CH = NOR ⁿ , -CH = NR ⁹ , -CH = NNHCH (N = NH) NH 2, -SO 2 NR ¹² R ¹³ , -PO (OR ⁿ ) 2 or OR ¹⁴ , where R ¹⁴ is an amino acid residue after the hydroxyl group of the carboxyl group is replaced, and or R ¹² and R ¹³ are each independently H, alkyl having from 1 to 4 carbons (including), aryl having from 6 to 10 carbon atoms, preferably phenyl or naphthyl, or heteroaryl; or R ¹² and R ¹³ together form a linking group, preferably - (CH ₂ ) 2-X ¹ - (CH 2) 2; each R ³ , R ⁴ , R ⁵ and R ⁶ is independently H, aryl, preferably aryl having from 6 to 10 carbon atoms (including), more preferably phenyl or naphthyl; heteroaryl, F, Cl, Br, I, -CN, CF3, -NO2, OH, -OR ⁹ , O (CH 2) 'NR ⁷ R ⁸ , -OCOR ⁹ , -OCONHR ⁹ , NH 2, -CH 2 OH, -CH ² OR ¹⁴ , -NR ⁷ R ⁸ , NR ¹⁰ COR ⁹ , -NR ¹⁰ CONR ⁷ R ⁸ , -SR ¹¹ , -S (O) y R ^u , where y is 1 or 2; -CO 2 R ⁹ , COR ⁹ , -CONR ⁷ R ⁸ , -CHO, -CH = NOR ⁿ , -CH = NR ⁹ , -CH = NNR ⁿ R ¹² , (CH 2) _n SR ⁹ , where n is an integer of 1 to 4 (inclusive), (CH 2) n S (O) y, R ⁹ , -CH 2 SR ¹⁵ , where R ¹⁵ is alkyl having from 1 to 4 carbon atoms (inclusive); -CH2S (O) yR ^14, - (CH2) NR ⁷ R ^8, - (CH _{2) n} NHR ^14, alkyl having from 1 to 8 carbons (inclusive), preferably alkyl having from 1 to 4 carbon atoms (including); alkenyl having from 1 to 8 carbon atoms (including), preferably alkenyl having from 1 to 4 carbon atoms (including); alkynyl having from 1 to 8 carbon atoms (including); preferably alkynyl having from 1 to 4 carbon atoms (including); and or any alkyl having from 1 to 8 carbons (including), alkenyl having from 1 to 8 carbons (including) or alkynyl having from 1 to 8 carbons (including) is unsubstituted; or any alkyl having from 1 to 8 carbons (incl.), alkenyl having from 1 to 8 carbons (incl.) or alkynyl having from 1 to 8 carbons (incl.) is substituted as described in d) (2), above; X is or unsubstituted alkylene having from 1 to 3 carbons (inclusive); or X is alkylene having from 1 to 3 carbons (inclusive), substituted by one R ² group, preferably OR ¹⁰ , -SR ¹⁰ , R ¹⁵ , where R ¹⁵ is alkyl having from 1 to 4 carbons (inclusive); phenyl, naphthyl, arylalkyl having from 7 to 14 carbons (inclusive), preferably benzyl; or X is -CH = CH-, -CH (OH) -CH (OH) -, -0-, -S-, -S (= O) -, S (= O) ₂ -, -CR ¹⁰ ) 2- , -C (= O) -, -C (= NOR ¹¹ ) -, -C (OR ⁿ ) (R ⁿ ) -, -C (= O) CHR ¹⁵ ) -, CHR ¹⁵ ) C (= O) - , -C (= NOR ^h ) CHR ¹⁵ ) -, -CHR ¹⁵ ) C (= NOR ^h ) -, -ch2z-, -z-ch ₂ , -CH ₂ ZCH ₂ -, where Z is, C (OR ⁿ ) (R ⁿ ), O, S, C (= O), C (= NOR ⁿ ), or NR ¹¹ ; or A ¹ and A ² together are each independently Η, Η; Η, -OR ¹¹ ; Η, -SR ¹¹ ; NR, NR ⁿ R ¹² ; go together represent = S or = NR ⁿ ; B ¹ and B ² together represent O; and each R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X is as defined above in c), d), e) and f), or A and A together represent O and B and B together are each independently Η, Η; H, -OR ¹¹ , H, -SR ¹¹ , H, -NR ^H R ¹² , or together represent = S or = NR ⁿ ; and each R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined above in c), d), e) and f). 74. The method of claim 73, wherein Ac A ₂ , R _b R ₃ and R 4 are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; RsnReca BCH ₃ ; and HCH ₂ . 75. The method of claim 73, wherein A _b A ₂ , R 1, R ₃ , R ₅ and Re are H; B [and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R 4 is Br and X is CH ₂ . 76. The method of claim 73, wherein A 1, A ₂ , R 1, R ₃ , R, and R ₆ are H; B] and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R4 is CH ₂ CH ₂ (2-Pyr); and X is CH ₂ . 77. The method of claim 73, wherein A _b L ₂ , R 1, R 3, R 5 and Re are Η; Bi and B ₂ together represent O; R ₂ is H; R 4 is CH ₂ CH ₂ (2-Pyrimidinyl); and X is CH ₂ . 78. The method of claim 73, wherein A _b A ₂ , R _b R ₃ , R ₅ and Re are H; B] and B ₂ together represent O; R ₂ is H; R4 is CH ₂ CH ₂ (2-Pyr); and X is CH ₂ . 79. The method of claim 73, wherein A _b A ₂ , R _b R ₂ , R ₃ , R ₅ and Re are H; Bi and B ₂ together represent O; R4 is CH ₂ CH ₂ (2-Pyridazinyl); and X is CH ₂ . 80. The method of claim 73, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and Re are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; and X is CH ₂ . 81. The method of claim 73, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and Re are H; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ CH ₂ OH; and X is CH ₂ . 82. The method of claim 73, wherein A _b A ₂ , R _b R ₂ , R ₃ , R 4, R ₅ and Re are H; Bi and B ₂ together represent O; and X is S. 83. The method of claim 73, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and R 6 are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ CH ₂ NHCO (4- (OH) Ph); and X is CH ₂ . 84. The method of claim 73, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and Re are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; and X is CH ₂ . 85. The method of claim 72, wherein the compound has the formula wherein ring B and ring F, independently and each together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring, where from 1 to 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring, where or 1) one carbon atom is replaced by an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced by sulfur and nitrogen atoms, oxygen and nitrogen atoms or two nitrogen atoms; or 3) three carbon atoms are replaced by three nitrogen atoms; R1 is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heteroarylalkyl; 6) -C (= O) R ⁹ , wherein R ⁹ is selected from the group consisting of alkyl, aryl and heteroaryl; b) -OR ¹⁰ , wherein R ¹⁰ is selected from the group consisting of H and alkyl having from 1 to 4 carbons; r) -C (= O) NH ₂ , -NR ^H R ¹² , - (CH2) pNR ⁿ R ¹² , - (CH2) _P OR ¹⁰ , O (CH2) POR ¹⁰ and -O (CH2) _p NR ⁿ R ¹² , wherein p is from 1 to 4; and where or 1) R ⁿ and R ¹² are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbons; or 2) R ⁿ and R ¹² together form a linking group of formula - (CH 2) 2-X '- (CH 2) 2-, wherein X ¹ is selected from the group consisting of 0-, -S- and -CH 2 -; R ² is selected from the group consisting of H, alkyl having from 1 to 4 carbons, -OH, alkoxy having from 1 to 4 carbons, -OC (= O) R ⁹ , OC (= O) NR ⁿ R ¹² , -O (CH2) pNR ⁿ R ¹² , -O (CH 2) _P OR ¹⁰ , substituted or unsubstituted arylalkyl having from 6 to 10 carbons and substituted or unsubstituted heteroarylalkyl; R ³ , R ⁴ , R ⁵ and R ⁶ are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF ₃ , -ΝΟ ₂ -, -OH, OR ⁹ , -O (CH 2) pNR ⁿ R ¹² , -OC (= O) R ⁹ , -OC (= O) NR ² R ⁷ , -OC (= O) NR ⁿ R ¹² , O (CH 2) _P OR ¹⁰ , -CH 2 OR ¹⁰ , -NR ⁿ R ¹² , -NR ¹⁰ S (= O) 2R ⁹ , -NR ¹⁰ C (= O) R ⁹ ; b) -CH ₂ OR ¹⁴ , wherein R ¹⁴ is an amino acid residue after the hydroxyl group of the carboxyl group is removed; B) -NR ¹⁰ C (= O) NR ⁿ R ¹² , -CO ² R ² , -C (= O) R ² , -C (= O) NR ⁿ R ¹² , CH = NOR ² , -CH = NR ⁹ , - (CH2) pNR ⁿ R ¹² , - (CH ₂ ) _P NHR ¹⁴ or -CH = NNR ² R ^2A , 2A. 2 where R is the same as R; r) -S (O) _y R ² - (CH 2) p S (O) _y R ⁹ , -CH 2 S (O) _y R ¹⁴ , where y is 0, 1 or 2; e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons and alkynyl having from 2 to 8 carbons, where 1) each alkyl, alkenyl or alkynyl group is unsubstituted; or 2) each alkyl, alkenyl or alkynyl group is substituted by from 1 to 3 groups selected from the group consisting of aryl having from 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy, hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy, alkoxy, alkoxy, alkoxy, alkoxy, alkoxy Cl, Br, I, -CN, -NO ₂ , -OH, -OR ⁹ , -X ² (CH 2) _p ^N R ⁿ R ¹² , X ² (CH 2) _p C (= O) NR ^1, R ¹² , -X ² (CH 2) pOC (= O) NR ⁿ R ¹² , -X ² (CH 2) _p CO ₂ R ⁹ , X ² (CH 2) _p S (O) y R ⁹ , -X ² (CH 2) _p NR ¹⁰ C (= O) NR ¹¹ R ¹² , -OC (= O) R ⁹ , -OCONHR ² , -O-tetrahydropyranyl, -NR ⁿ R ¹² , -NR ¹⁰ C (= O) R ⁹ , -NR ¹⁰ CO2R ⁹ , NR ¹⁰ C (= O) NR ⁿ R ¹² , -NHC (= NH) NH 2, NR ¹⁰ S (O) 2 R ⁹ , -S (O) y R ⁹ , -CO ₂ R ⁹ , -C (= O) NR ⁿ R ¹² , -C (= O) R ² , -CH ² OR ¹⁰ , -CH = NNR ² R ^2A , CH = NOR ² , -CH = NR ⁹ , - CH = NNHCH (N = NH) NH2, -S (= O) ₂ NR ² R ^2A , P (= O) (OR ¹⁰ ) ₂ , -OR ¹⁴ and a monosaccharide having from 5 to 7 carbons, where each hydroxyl group the monosaccharide is independently or unsubstituted or substituted by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons; X ² is O, S or NR ¹⁰ ; R ⁷ and R ⁸ are each independently selected from the group consisting of H, alkyl having from 1 to 4 carbons, alkoxy having from 1 to 4 carbons, substituted or unsubstituted arylalkyl having from 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl, - (CH 2) pOR ¹⁰ , (CH 2) _p OC (= O) NR ¹¹ R ¹² and - (CH 2) _p NR ^{12 n} R ¹² , or R ⁷ and R ⁸ together form a linking group of the formula -CH 2 -X-CH ₂ - where X is X or a bond; m and η are independently 0, 1 or 2; Υ is selected from the group consisting of -O-, -S-, -N (R ¹⁰ ) -, -N ⁺ (O ') (R ¹⁰ ) -, N (OR ¹⁰ ) - and -CH ₂ -; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, C (= O) -, -CH (OR ¹⁰ ), -N (R ¹⁰ ) -, -N (OR ¹⁰ ) -, -CH (NR ^U R ¹² ) -, -C (= O) N (R ¹⁷ ) -, N (R ¹⁷ ) C (= O) -, -N (S (O) y R ⁹ ) -, -N (S (O) yNR ⁿ R ¹² ) -, -N (C (= O) R ¹⁷ ) -, C (R ¹⁵ R ¹⁶ ) -, -N ⁺ (O) (R ¹⁰ ) -, -CH (OH) -CH (OH) - and CH (O (C = O) R ⁹ ) CH (OC (= O) R ^9A ) -, where R ^9A is the same as R ⁹ ; R ¹⁵ and R ¹⁶ are independently selected from the group consisting of H, -OH, C (= O) R ¹⁰ , -O (C ^: = O) R ⁹ , hydroxyalkyl and -CO ₂ R ¹⁰ ; R ¹⁷ is selected from the group consisting of H, alkyl, aryl and heteroaryl; A ¹ and A ² are independently selected from the group consisting of Η, H; H, OR ² ; H, -SR ² ; H, N (R ² ) ₂ ; and a group wherein A ¹ and A ² taken together form a unit selected from the group containing = O, = S and = NR; B ¹ and B ² are selected from the group consisting of Η, Η; H, -OR ² ; H, SR ² , H, -N (R ² ) ₂ ; and a group wherein B ¹ and B ² together form a unit selected from the group containing = O, = S and = NR; Provided that at least one of the pairs A and A, or B and B, form = 0. 86. The method of claim 72, wherein the compound has the formula wherein Z 1 is H and Z ₂ is H or Z 1 and Z ₂ together form = O; R 1 is selected from the group consisting of H, Cl, CH ₂ SO ₂ C ₂ H ₅ , Br, CH ₂ S (CH ₂ ) ₂ NH ₂ , CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH ₂ , P-C 4 H 9, NHCONHC ₆ H ₅ , NHCONHC ₂ H _s , CH ₂ SC ₂ H ₅ , CH ₂ SC ₆ H ₅ , N (CH ₃ ) ₂ , CH3, CH ₂ OCONHC ₂ H ₅ , NHCO ₂ CH ₃ , CH ₂ OC ₂ H ₅ , CH ₂ N (CH ₃ ) ₂ , OH, 0 n-propyl, CH = NNH-C (= NH) NH ₂ , CH = NN (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH-nC ₄ H ₉ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 [3- (1,2,4-triazine)], CH ₂ CH ₂ SCH _3> / ~ Ά Ν Ο C№ = N— ΛΑ CH — Ν — Ν NCH3 \ _7 And R ₂ is selected from the group consisting of H, Br, Cl, I, CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 (3- (1,2,4-triazine)), CH ₂ CH ₂ SCH ₃ and CH ₂ OH; X is selected from the group consisting of H, CH ₂ OH, CH ₂ L ₁ H-Serine H, CO ₂ CH ₃ , CONHC ₆ H ₅ , ch ₂ nhco ₂ c ₆ h ₅ , ch ₂ nhco ₂ ch ₃ , ch ₂ n ₃ , CONHC ₂ H ₅ , CH 2 H-Glycine, CON (CH ₃ ) ₂ , -CH ₂ NHCO ₂ -, conh ₂ , CONHC ₃ H ₇ , CH ₂ NH-CepHH, CH ₂ SOCH ₃ , CH = NOH. CH ₂ NH-11rolin, CH ₂ CH ₂ (2-Pyridyl), CH = NNHC (= NH) NH ₂ , CONH (CH ₂ ) ₂ OH, CH = NNHCONH ₂ , CH ₂ OCOCH ₃ , -CH ₂ OC (CH ₃ ) ₂ O-, ch ₂ sc ₆ h ₅ , CH ₂ SOC ₆ H ₅ , CO ₂ n-hexyl, CONHCH ₃ and CO ₂ (CH ₂ ) ₄ CH ₃ ; and R is selected from the group consisting of OH and OCH ₃ . 87. The method of claim 86, wherein Ζι and Z ₂ are Η; X is CO ₂ CH ₃ ; R1 is NHCONHC ₂ H ₅ ; R ₂ is CH ₂ CH ₂ (2-Pyridyl); and R is OH. 88. The method of claim 86, wherein Ζι and Z ₂ are Η; X is CO ₂ CH ₃ ; R 1 and R ₂ are CH ₂ OCH ₂ OCH ₂ CH ₃ ; and R is OH. 89. The method of claim 86, wherein Ζι and Ζ ₂ are Η; X is CO ₂ CH ₃ ; R 1 and R ₂ are CH ₂ SCH ₂ CH ₃ ; and R is OH. 90. The method of claim 86, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ and R is OH. 91. The method of claim 86, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ (CH ₂ ) ₄ CH ₃ ; and R is OH. 92. The method of claim 86, wherein Z _b Z ₂ and R _b are H; R ₂ is CH ₂ OH; X is CO ₂ CH ₃ ; and R is OH. 93. The method of claim 86, wherein Ζι and Ζ ₂ are H; R 1 and R ₂ are H ₂ 8 (3- (1,2,4-triazine)); X is CO ₂ CH ₃ ; and R is OH. 94. The method of claim 86, wherein Ζι and Ζ ₂ are H; R1 is Br; R ₂ is I; X is CO ₂ CH ₃ ; and R is OH. 95. The method of claim 86, wherein Ζι and Ζ ₂ are H; R 1 and R ₂ are CH ₂ CH ₂ SCH ₃ ; X is CO ₂ CH ₃ ; and R is OH. 96. The method of claim 86, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ ; and R is OCH ₃ . 97. The method of claim 86, characterized in that Ζ] and Ζ ₂ together form O; R 1 and R ₂ are Br; X is CO ₂ CH ₃ ; and R is OH. The method of claim 72, wherein the compound has the formula wherein: Zi is H and Z ₂ is H or Ζι and Z ₂ together form = 0; R1 is H or Br; R ₂ eH; j__ R ₃ is H, CH ₂ CH = CH ₂ , CH ₂ CH ₂ CH ₂ OH, or CH ₂ CH ₂ CH ₂ - __O and R4 is H, CH ₂ CH = CH ₂ or CH ₂ CH ₂ CH ₂ OH. 99, The method of claim 98, wherein R _b R ₂ , R 4, Z ₂ and Z ₂ are H and R ₃ is CH ₂ CH = CH ₂ . The method of claim 98, wherein R 1 is Br and R ₂ , R ₃ R 4, Ζι and Z ₂ are Η. ϊ 101. The method of claim 98, wherein R _b R ₂ , Ζι and Z ₂ are H and R ₃ and R 4 are CH ₂ CH = CH ₂ . 102. The method of claim 98, wherein R1, R ₂ , R ₃ , Z 1 and Z ₂ are H and R 4 is CH ₂ CH = CH ₂ . 103. The method of claim 98, wherein R 1, R ₂ , Z 1 and Z ₂ are H, and R ₃ and R 4 are CH ₂ CH ₂ CHOH; or R _b R ₂ , R 4, Ζι and Z ₂ are Η, CH2CH2CH2 —__ D 104. A method of treating mammals having inflammation comprising administering to a mammal a compound that inhibits a complex kinase protein line in a pharmaceutically acceptable salt or diluent. 105. The method of claim 104, wherein the compound has the formula wherein E ¹ and E ² , independently and each together with the carbon atoms to which they are attached, form either an unsaturated 6-membered carbocyclic aromatic ring in which one to three carbon atoms may be replaced by a nitrogen atom (s) ); or an unsaturated 5-membered carbocyclic aromatic ring; wherein either one carbon atom is replaced by an oxygen, nitrogen or sulfur atom; or two carbon atoms are replaced by a sulfur and nitrogen atom, or an oxygen and nitrogen atom; A ¹ and A ² together represent 0 and B ¹ and B ² together represent 0; R ¹ is H, alkyl having from 1 to 4 carbon atoms (including), aryl, arylalkyl, heteroaryl and heteroarylalkyl; COR ⁹ , wherein R ⁹ is alkyl having from 1 to 4 carbon atoms (including) or aryl, preferably phenyl or naphthyl; -OR ¹⁰ , wherein R ¹⁰ is H or alkyl having from 1 to 4 carbon atoms (including); -CONH2, -NR R, (CH2) nNR ⁷ R ⁸ , where n is an integer from 1 to 4 (inclusive); or O (CH 2) 'NR ⁷ R ⁸ and or R ⁷ and R ^{8 are} independently H or alkyl having from 1 to 4 carbon atoms (including), or R ⁷ and R ⁸ are joined together to form a linking group of the general formula - (CH 2 - 1 -C 3 H 2 - -, wherein X ¹ is O, S or CH ₂ ; R ² is H, -SO ² R ⁹ ; -CO ² R ⁹ , -COR ⁹ , alkyl having from 1 to 8 carbon atoms (including), preferably alkyl having from 1 to 4 carbon atoms (including), alkenyl having from 1 to 8 carbon atoms (including) , preferably alkenyl having from 1 to 4 carbon atoms (including), or alkynyl having from 1 to 8 carbon atoms (including), preferably alkynyl having from 1 to 4 carbon atoms (including), or a monosaccharide having from 5 to 7 carbon atoms (inclusive), wherein each hydroxyl group of the monosaccharide is independently or unsubstituted or replaced by H, alkyl having from 1 to 4 carbon atoms (including), alkylcarbonyloxy having from 2 to 5 carbon atoms (including) or an alkoxy group having from 1 to 4 carbon atoms (including), and or any alkyl having from 1 to 8 carbon atoms ( alkenyl having from 1 to 8 carbon atoms (including), or alkynyl having from 1 to 8 carbon atoms (including) is unsubstituted; or each alkyl having from 1 to 8 carbon atoms (including), alkenyl having from 1 to 8 carbon atoms (including), or alkynyl having from 1 to 8 carbon atoms (including) is independently substituted by 1-3 aryl having from 6 to 10 carbon atoms (including), preferably phenyl or naphthyl, heteroaryl, F, Cl, Br, I, -CN, -NO ₂ , OH, -OR ⁹ , -O (CH 2) n NR ⁷ R ⁸ , -OCOR ⁹ , -OCONHR ⁹ , O-tetrahydropyranyl, NH 2, -NR ⁷ R ⁸ , -NR ¹⁰ COR ⁹ ; -NR ¹⁰ CO2R ⁹ , -NR ¹⁰ CONR ⁷ R ⁸ , -NHC (= NH) NH 2, - NR ¹⁰ SO ² R ⁹ , -S (O) y R ⁿ , where R ¹¹ is H or alkyl having from 1 to 4 carbon an atom, an aryl having from 6 to 10 carbon atoms, preferably phenyl or naphthyl or heteroaryl and y is 1 or 2; -SR ¹¹ , CO2R ⁹ , -CONR ⁷ R ⁸ , -CHO, COR ⁹ , -CH2OR ⁷ , -CH = NNR ⁿ R ¹² , -CH = NOR ⁿ , -CH = NR ⁹ , -CH = NNHCH (N = NH) NH2, -SO2NR ¹² R ¹³ , -PO (OR ⁿ ) 2 or OR ¹⁴ , where R ¹⁴ is an amino acid residue after the hydroxyl group of the carboxyl group is replaced, and or R ¹² and R ¹³ are each independently H, alkyl having from 1 to 4 carbon atoms (including), aryl having from 6 to 10 carbon atoms, preferably phenyl or naphthyl, or heteroaryl; or R ¹² and R ¹³ together form a linking group, preferably - (CH 2) 2 -X ¹ - (CH ₂ ) ₂ ; each R ³ , R ⁴ , R ⁵ and R ⁶ is independently H, aryl, preferably aryl having from 6 to 10 carbon atoms (including), more preferably phenyl or naphthyl; heteroaryl, F, Cl, Br, I, -CN, CF3, -NO2, OH, -OR ⁹ , O (CH 2) _n NR ⁷ R ⁸ , -OCOR ⁹ , -OCONHR ⁹ , NH 2, -CH 2 OH, -CH ² OR ¹⁴ , -NR ⁷ R ⁸ , NR ¹⁰ COR ⁹ , -NR ¹⁰ CONR ⁷ R ⁸ , -SR ¹¹ , -S ^ R ¹¹ , where y is 1 or 2; -CO2R ⁹ , COR ⁹ , -CONR ⁷ R ⁸ , -CHO, -CH = NOR ⁿ , -CH = NR ⁹ , -CH = NNR ⁿ R ¹² , (CH2) nSR ⁹ , where n is an integer from 1 to 4 (inclusive), (CH 2) _n S (O) _y , R ⁹ , -CH 2 SR ¹⁵ , wherein R ¹⁵ is alkyl having from 1 to 4 carbon atoms (inclusive); -CH2S (O) _y R ^14, - (CH2) NR ⁷ R ^8, - (CH2) _n NHR ^14, alkyl having from 1 to 8 carbons (inclusive), preferably alkyl having from 1 to 4 carbon atoms (including); alkenyl having from 1 to 8 carbon atoms (including), preferably alkenyl having from 1 to 4 carbon atoms (including); alkynyl having from 1 to 8 carbon atoms (incl.); preferably alkynyl having from 1 to 4 carbon atoms (including); and or each alkyl having from 1 to 8 carbon atoms (including), alkenyl having from 1 to 8 carbon atoms (including) or alkynyl having from 1 to 8 carbon atoms (including) is unsubstituted; or any alkyl having from 1 to 8 carbon atoms (including), alkenyl having from 1 to 8 carbon atoms (including) or alkynyl having from 1 to 8 carbon atoms (including) is substituted as described in d) 2), above; X is or unsubstituted alkylene having from 1 to 3 carbon atoms (including); or X is alkylene having from 1 to 3 carbon atoms (including), substituted by one R ² group, preferably OR ¹⁰ , -SR ¹⁰ , R ¹⁵ , where R ¹⁵ is alkyl having from 1 to 4 carbon atoms (including) ; phenyl, naphthyl, arylalkyl having from 7 to 14 carbon atoms (including), preferably benzyl; or X is -CH = CH-, -CH (OH) -CH (OH) -, -0-, -S-, -S (= O) -, S (= O) ₂ -, -CR ¹⁰ ) 2- , -C (= O) -, -C (= NOR ⁿ ) -, -C (OR ¹¹ ) (R ¹¹ ) -, -C (= O) CHR ¹⁵ ) -, CHR ^I5 ) C (= O) - , -C (= NOR ⁿ ) CHR ¹⁵ ) -, -CHR ¹⁵ ) C (= NOR ¹¹ ) -, -CH ₂ Z-, -Z-CH ₂ , -CH ₂ ZCH ₂ -, where Z is C (OR ⁿ ) (R ⁿ ), O, S, C (-O), C (= NOR ⁿ ), or NR ¹¹ ; or A ¹ and A ² together are each independently Η, Η; Η, -OR ¹¹ ; Η, -SR ¹¹ ; NR, NR ⁿ R ¹² ; or together represent = S or = NR ⁿ ; B ¹ and B ² together represent O; and each R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X is as defined above in c), d), e) and f) or A ¹ and A ² together represent O and B ¹ and B ² together are each independently Η, Η; H, -OR ¹¹ , H, -SR ¹¹ , H, -NR ⁿ R ¹² , or together represent = S or = NR ⁿ ; and each R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined above in c), d), e) and f). The method of claim 105, wherein Ac ₂ , R 1, R ₃ and R 4 are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; R ₅ and R ₅ are OCH ₃ ; and X is CH ₂ . The method of claim 105, wherein A _b A ₂ , R 1, R ₃ , R ₅ and R ₆ are H; B and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R4 is Br; and X is CH ₂ . 108. The method of claim 105, wherein A _b A ₂ , R _b R ₃ , R 5 and R ₆ are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OAc; R4 is CH ₂ CH ₂ (2-Pyr); and X is CH ₂ . 109, The method of claim 105, wherein A, A ₂ , R _b R ₃ , R ₅ and Re are H; Bj and B ₂ together represent O; R ₂ is H; R 4 is CH ₂ CH ₂ (2-Pyrimidinyl); and X is CH ₂ . The method of claim 105, wherein A _b A ₂ , R _b R ₃ , R ₅ and R ₆ are Η; Bi and B ₂ together represent O; R ₂ is H; R4 is CH ₂ CH ₂ (2-Pyr); and X is CH ₂ . 111. The method of claim 105, wherein A _b A ₂ , R _b R ₂ , R ₃ , R ₅ and R ₆ are H; Bj and B ₂ together represent O; R4 is CH ₂ CH ₂ (2-Pyridazinyl); and X is CH ₂ . 112. The method of claim 105, wherein A _b A ₂ , R 1, R ₃ , R 4, R 5 and Re are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; and X is CH ₂ . The method of claim 105, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and Re are H; B _t and B ₂ together represent O; R ₂ is CH ₂ CH ₂ CH ₂ OH; and X is CH ₂ . The method of claim 105, wherein A _b A ₂ , R _b R ₂ , R ₃ , R 4, R ₅ and Re are H; B 1 and B ₂ together represent O; and X is S. 115. The method of claim 105, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and Re are Η; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ CH ₂ NHCO (4- (OH) Ph); and X is CH ₂ . 116. The method of claim 105, wherein A _b A ₂ , R _b R ₃ , R 4, R ₅ and Re are H; Bi and B ₂ together represent O; R ₂ is CH ₂ CH ₂ OH; and X is CH ₂ . 117. The method of claim 104, wherein the compound has the formula Yal And where: ring B and ring F, independently of each and together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring, where from 1 to 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring, where or 1) one carbon atom is replaced by an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced by sulfur and nitrogen atoms, oxygen and nitrogen atoms or two nitrogen atoms; and 3) three carbon atoms are replaced by three nitrogen atoms; R ¹ is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heteroarylalkyl; 6) -C (= O) R ⁹ , wherein R ⁹ is selected from the group consisting of alkyl, aryl and heteroaryl; b) -OR ¹⁰ , wherein R ¹⁰ is selected from the group consisting of H and alkyl having from 1 to 4 carbon atoms; r) -C (= O) NH ₂ , -NR ⁿ R ¹² , - (CH2) pNR ⁿ R ¹² , - (CH2) _P OR ¹⁰ , O (CH2) pOR ¹⁰ and -O (CH2) _p NR ⁿ R ¹² , wherein p is from 1 to 4; and where or 1) R ⁿ and R ¹² are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbon atoms; or 2) R ^H and R ¹² together form a linking group of formula - (CH 2) 2-X ¹ - (CH ₂ ) ₂ -, wherein X ¹ is selected from the group consisting of 0-, -S- and -CH ₂ _; R ² is selected from the group consisting of H, alkyl having from 1 to 4 carbon atoms, -OH, alkoxy having from 1 to 4 carbon atoms, 33 OC (= O) R ^{9, -OC (= O) NR n} R ^{12, -O (CH2) pNR n R} i2, -O (CH2) P OR ^10, substituted or unsubstituted arylalkyl having from 6 to 10 carbon atoms and substituted or unsubstituted heteroarylalkyl; R ³ , R ⁴ , R ⁵ and R ⁶ are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF ₃ , -ΝΟ ₂ -, -OH, OR ⁹ , -O (CH 2) pNR ⁿ R ¹² , -OC (= O) R ⁹ , -OC (= O) NR ² R ⁷ , -OC (= O) NR ⁿ R ¹² , O (CH 2) _P OR ¹⁰ , -CH 2 OR ¹⁰ , -NR ⁿ R ¹² , -NR ¹⁰ S (= O) 2R ⁹ , -NR ¹⁰ C (= O) R ⁹ ; 6) -CH ₂ OR ¹⁴ , wherein R ¹⁴ is an amino acid residue after the hydroxyl group of the carboxyl group is removed; b) -NR ¹⁰ C (= O) NR ^h R ¹² , -CO ² R ² , -C (= O) R ² , -C (= O) NR ⁿ R ¹² , CH = NOR ² , -CH = NR ⁹ , - (CH2) _p NR '' r ' ² , - (CH ₂ ) _p NHR ¹⁴ or -CH = NNR ² R ^2A , where R ^2a is the same as R ² ; r) -S (O) _y R ² - (CH 2) p S (O) _y R ⁹ , -CH 2 S (O) _y R ¹⁴ , where y is 0, 1 or 2; e) alkyl having from 1 to 8 carbon atoms, alkenyl having from 2 to 8 carbon atoms and alkynyl having from 2 to 8 carbon atoms, where 1) each alkyl, alkenyl or alkynyl group is unsubstituted; or 2) each alkyl, alkenyl or alkynyl group is substituted by from 1 to 3 groups selected from the group consisting of aryl having from 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy, hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy, alkoxy, alkoxy, alkoxy, alkoxy, alkoxy Cl, Br, I, -CN, -NO ₂ , -OH, -OR ⁹ , -X ² (CH 2) pNR ⁿ R ¹² , X ² (CH 2) _p C (= O) NR ⁿ R ¹² , -X ² (CH2) pOC (= O) NR ⁿ R ¹² , -X ² (CH 2) _P CO ₂ R ⁹ , X ² (CH 2) _p S (O) y R ⁹ , -X ² (CH 2) _p NR ¹⁰ C (= O ) NR ¹¹ R ¹² , -OC (= O) R ⁹ , -OCONHR ² , O-tetrahydropyranyl, -NR ⁿ R ¹² , -NR ¹⁰ C (= O) R ⁹ , -NR ¹⁰ CO ₂ R ⁹ , NR ¹⁰ C (= O) NR ⁿ R ¹² , -NHC (= NH) NH ₂ , NR ¹⁰ S (O) 2 R ⁹ , -S (O) y R ⁹ , -CO ₂ R ⁹ , C (= O) NR ⁿ R ¹² , -C (= O) R ² , -CH ² OR ¹⁰ , -CH = NNR ² R ^2A , -CH = NOR ² , CH = NR ⁹ , -CH = NNHCH (N = NH) NH 2, -S ( = O) ₂ NR ² R ^2A , -P (= O) (OR ¹⁰ ) ₂ , OR ¹⁴ and a monosaccharide having from 5 to 7 carbon atoms, where each hydroxyl group of the monosaccharide is independently or unsubstituted or 3 substituted by H, alkyl having from 1 to 4 carbon atoms, alkylcarbonyloxy having from 2 to 5 carbon atoms, or alkoxy having from 1 to 4 carbons; X ² is O, S or NR ¹⁰ ; R ⁷ and R ⁸ are each independently selected from the group consisting of H, alkyl having from 1 to 4 carbon atoms, alkoxy having from 1 to 4 carbon atoms, substituted or unsubstituted arylalkyl having from 6 to 10 carbon atoms, substituted or unsubstituted heteroarylalkyl, (CH 2) pOR ¹⁰ , - (CH 2) _p OC (= O) NR ¹¹ R ¹² and - (CH 2 pNR ¹¹ ^ ² , or R ⁷ and R ⁸ together form a linking group of formula -CH ₂ -X -CH ₂ -, wherein X is X or a bond; y and η are independently 0, 1 or 2; Υ is selected from the group consisting of -O-, -S-, -N (R ¹⁰ ) -, -N ⁺ (O ') (R ¹⁰ ) -, N (OR ¹⁰ ) - and -CH ₂ -; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, C (= O) -, -CH (OR ¹⁰ ), -N (R ¹⁰ ) -, -N (OR ¹⁰ ) -, -CH (NR ⁿ R ¹² ) -, -C (O) N (R ¹⁷ ) -. N (R ¹⁷ ) C (= O) -, -N (S (O) yR ⁹ ) -, -N (S (O) yNR ⁿ R ¹² ) -, -N (C (= O) R ¹⁷ ) - , C (R ¹⁵ R ¹⁶ ) -, -N ⁺ (O ') (R ¹⁰ ) -, -CH (OH) -CH (OH) -, and CH (O (C = O) R ⁹ ) CH (OC ( = O) R ^9A ) - wherein R ^9A is the same as R ⁹ ; R ¹⁵ and R ¹⁶ are independently selected from the group consisting of H, -OH, C (= O) R ¹⁰ , -O (CO) R ⁹ , hydroxyalkyl and -CO ₂ R ¹⁰ ; R ¹⁷ is selected from the group consisting of H, alkyl, aryl and heteroaryl; A ¹ and A ² are independently selected from the group consisting of Η, Η; H, OR ² ; H, -SR ² ; H, -N (R ² ) ₂ ; and a group wherein A ¹ and A ² taken together form a unit selected from the group containing = O, = S and = NR; 12 2 2 B and B are selected from the group consisting of Η, Η; H, -OR; H, -SR, 9 1 about H, -N (R) ₂ ; and a group wherein B and B together form a unit selected from the group containing = O, = S and = NR; Provided that at least one of the pairs A and A, or B and B, form = 0. The method of claim 104, wherein the compound has the formula wherein Z] is H and Z ₂ is H or Ζι and Ζ ₂ together form = 0; R 1 is selected from the group consisting of H, Cl, CH ₂ SO ₂ C ₂ H ₅ , Br, CH ₂ S (CH ₂ ) ₂ NH ₂ , CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH ₂ , P-C 4 H 9, nhconhc ₆ h ₅ , nhconhc ₂ h ₅ , CH ₂ SC ₂ H ₅ , CH ₂ SC ₆ H ₅ , N (CH ₃ ) ₂ , ch ₃ , CH ₂ OCONHC ₂ H ₅ , NHCO ₂ CH ₃ , CH ₂ OC ₂ H ₅ , CH ₂ N (CH ₃ ) ₂ , OH, Ο n-propyl, CH = NNH-C (= NH) NH ₂ , CH = NN ( CH ₃ ) ₂ , CH ₂ S (CH ₂ ) ₂ NH-nC ₄ H ₉ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ S (3- (1,2,4-triazine)), CH ₂ CH ₂ SCH ₃ , / \ CH—: N— Q Γ ~ \ CH = N — N NCHj w R ₂ is selected from the group consisting of H, Br, Cl, I, CH ₂ S (CH ₂ ) ₂ N (CH ₃ ) ₂ , NHCONHC ₂ H ₅ , CH ₂ SC ₂ H ₅ , CH ₂ OCH ₂ OCH ₂ CH ₃ , CH ₂ 8 (3- (1,2,4-triazine)), CH ₂ CH ₂ SCH ₃ and CH ₂ OH; X is selected from the group consisting of H, CH ₂ OH, CH ₂ CN-SerineH, CO ₂ CH ₃ , CONHC ₆ H ₅ , CH ₂ NHCO ₂ C ₆ H ₅ , ch ₂ nhco ₂ ch ₃ , ch ₂ n ₃ , CONHC ₂ H ₅ , CH 2 H-Glycine, CON (CH ₃ ) ₂ , -CH ₂ NHCO ₂ -, conh ₂ , CONHC ₃ H ₇ , CH ₂ M1-Serine, CH ₂ SOCH ₃ , CH = NOH. CH ₂ ES-Proline, CH ₂ CH ₂ (2-Pyridyl), CH = NNHC (= NH) NH ₂ , CONH (CH ₂ ) ₂ OH, CH = NNHCONH ₂ , CH ₂ OCOCH ₃ , -CH ₂ OC (CH ₃ ) ₂ O-, ch ₂ sc ₆ h ₅ , CH ₂ SOC ₆ H ₅ , CO ₂ n-hexyl, CONHCH ₃ , CO ₂ (CH ₂ ) ₄ CH ₃ ; and R is selected from the group consisting of OH and OCH ₃ . 119. The method of claim 118, wherein Zj and Z ₂ is Η; X is CO ₂ CH ₃ ; R1 is NHCONHC ₂ H ₅ ; R ₂ is CH ₂ CH ₂ (2-Pyridyl); and R is OH. 120. The method of claim 118, wherein Ζι and Z ₂ are Η; X is CO ₂ CH ₃ ; R! and R ₂ is CH ₂ OCH ₂ OCH ₂ CH ₃ ; and R is OH. 121. The method of claim 118, wherein Ζι and Z ₂ are Η; X is CO ₂ CH ₃ ; R 1 and R ₂ are CH ₂ SCH ₂ CH ₃ ; and R is OH. 122. The method of claim 118, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ ; and R is OH. 123. The method of claim 118, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ (CH ₂ ) ₄ CH ₃ ; and R is OH. 124. The method of claim 118, wherein Z _b Z ₂ and R _b are H; R ₂ is CH ₂ OH; X is CO ₂ CH ₃ ; and R is OH. 125. The method of claim 118, wherein Z ₂ and Z ₂ are H; R 1 and R ₂ are H ₂ 8 (3- (1,2,4-triazine)); X is CO ₂ CH ₃ ; and R is OH. 126. The method of claim 118, wherein Z _t and Z ₂ are H; R1 is Br; R ₂ is I; X is CO ₂ CH ₃ ; and R is OH. 127. The method of claim 118, wherein Ζι and Ζ ₂ are H; R 1 and R ₂ are CH ₂ CH ₂ SCH ₃ ; X is CO ₂ CH ₃ ; and R is OH. 128. The method of claim 118, wherein Z _b Z ₂ , R 1 and R ₂ are Η; X is CO ₂ CH ₃ ; and R is OCH ₃ . 129. The method of claim 118, wherein Ζ, and Ζ ₂ together form = 0; R 1 and R ₂ are Br; X is CO ₂ CH ₃ ; and R is OH. 130. The method of claim 104, wherein a compound has the formula wherein: is H and Z ₂ is H or илиι and Ζ ₂ together form = 0; R1 is H or Br R ₂ eH; R ₃ is H, CH ₂ CH = CH ₂ , CH ₂ CH ₂ CH ₂ OH or CH ₂ CH ₂ CH ₂ - R4 is H, CH ₂ CH = CH ₂ or CH ₂ CH ₂ CH ₂ OH. The method of claim 130, wherein R _b R ₂ , R 4, Z 1 and Z ₂ are H and R ₃ is CH ₂ CH = CH ₂ . The method of claim 130, wherein R 1 is Br and R ₂ , R ₃ , R 4, Z 1 and Z ₂ are Η. The method of claim 130, wherein R _b R ₂ , Z 1 and Z ₂ are H, and R ₃ and R 4 are CH ₂ CH = CH ₂ . The method of claim 130, wherein R _b R ₂ , R ₃ , Z 1 and Z ₂ are H, and R 4 is CH ₂ CH = CH ₂ . 135. The method of claim 130, wherein R _b R ₂ , Z 1 and Z ₂ are H and R ₃ and R 4 are CH ₂ CH ₂ CH ₂ OH; or R _b R ₂ , R 4, Ζχ and Ζ ₂ are